Astudy ofthe intracellular signalling events involved ...

A study of the intracellular signallingevents involved in the zona pellucida-induced acrosome reaction in human

spermatozoa

by

SIMON STEPHANUS DU PLESSIS

Dissertation presented for the degree of

Doctor of Philosophy

at the Faculty of Health Sciences, University of Stellenbosch

Promotors:

Prof DR Franken and Dr C Page

December 2002

ii

DECLARA TION

I, the undersigned, hereby declare that the work in this dissertation is my own original

work and that I have not previously in its entirety or in part submitted it at any

university for a degree.

Dal

Stellenbosch University http://scholar.sun.ac.za

iii

ABSTRACT

In this study the author presents new data that will shed light on the fairly nebulous

knowledge of intracellular pathways involved in the physiologically induced acrosome

reaction and the subsequent events leading to fertilization. The zona pellucida-

induced acrosome reaction, sperm-zona interaction as well as various sperm motion

characteristics were investigated.

The first part of the study focussed on the role of extracellular signal regulated kinase

(ERK), a member of the family of mitogen activated protein kinases, during the zona

pellucida-induced acrosome reaction and sperm-oocyte interaction. It was shown that

the inhibition of ERK significantly reduced the zona pellucida-induced acrosome

reaction as measured by fluorescent microscopy. This suggests that ERKs are

directly or indirectly involved in the signal transduction pathway through which the

human sperm acrosome reaction is induced by the zona pellucida. In an attempt to

provide further proof that ERK was involved in human acrosome signalling hemizona

assays were employed to test sperm-oocyte binding. From these sperm-oocyte-

binding experiments it was clear that the inhibition of ERK leads to increased binding.

These results support the idea that the zona pellucida-induced acrosome reaction,

and therefore the physiologically relevant exocytotic event, is regulated by an ERK-

mediated signal transduction process.

In the second part of the study the significance of phosphatidylinositol 3-kinase

(PI3K) in the process of human sperm motility, acrosome reaction and sperm-oocyte

binding, was investigated by employing the specific PI3K, LY294002. PI3K inhibition

increased the percentage motility and percentage progressive motility in


iv

asthenozoospermia patients. Under the present laboratory conditions PI3K inhibition

furthermore did not influence the acrosome reaction, whilst it enhanced sperm-oocyte

binding. These results therefore imply that PI3K negatively affect sperm motility and

zona-binding.

In the last part of the study the possible effects of intracellular cGMP accumulation

via acute in vivo sildenafil citrate (ViagraTM) administration was investigated on

seminal parameters, induction of the acrosome reaction, sperm-oocyte binding and

sperm motility. As was expected no changes in the macro- and microscopically

seminal parameters were caused by sildenafil citrate when compared to placebo.

Furthermore the acrosome reaction was also not initiated or potentiated by sildenafil

citrate at concentrations of 50mg orally. Sperm-oocyte binding, smooth path velocity,

straight line velocity and the percentage rapid cells all increased after sildenafil citrate

treatment.

From these results it appear that there are various role players in the zona pellucida-

induced acrosome reaction intracellular signalling system. A thorough understanding

of such signal transduction systems and the crosstalk in-between will ultimately yield

information regarding the nature of receptors to which these signal transduction

pathways are coupled in human spermatozoa as well as the intracellular effectors

that ultimately regulate sperm function. Moreover, an understanding of these

regulatory pathways will be essential for the future development of clinical

approaches designed to enhance or preclude fertilization.


v

OPSOMMING

Die outeur lê in hierdie studie nuwe data voor ten einde meer lig te werp op die

relatief vae veld van intrasellulêre seintransduksie paaie betrokke by die fisiologies-

geïnduseerde akrosoomreaksie en die daaropvolgende gebeure wat aanleiding gee

tot bevrugting. Die zona pellucida-geïnduseerde akrosoomreaksie, sperm-zona

interaksie sowel as spermmotiliteitseienskappe is ondersoek.

Die eerste gedeelte van die studie fokus op die rol van ekstrasellulêre-

seingereguleerde-kinase (ERK), 'n lid van die familie van mitogeen-geaktiveerde

proteïenkinases, tydens die zona pellucida-geïnduseerde akrosoomreaksie en

sperm-oosiet interaksie. Daar word aangetoon dat die inhibisie van ERK die zona

pellucida geïnduseerde akrosoomreaksie, soos gemeet met behulp van fluorosensie

mikroskopie, betekenisvol verminder. Dit suggereer dat ERK direk of indirek betrokke

is by die seintransduksie paaie waardeur die akrosoomreaksie van die menslike

sperm deur die zona pellucida geïnduseer word. In 'n poging om onomwonde te

bewys dat ERK betrokke is by menslike akrosoom-seintransduksie, is hemizona

essais gebruik om sperm-oesiet binding te bepaal. Van hierdie sperm-oosiet binding-

eksperimente is dit duidelik dat die inhibisie van ERK aanleiding gee tot verhoogde

binding. Hierdie resultate ondersteun dus die idee dat die zona pellucida-

geïnduseerde akrosoomreaksie en dus die fisiologies relevante eksositotiese

gebeurtenis gereguleer word deur 'n ERK-gemediëerde seintransduksie proses.

In die tweede gedeelte van die studie is die belang van fosfatidielinositol 3-kinase

(PI3K) in die proses van menslike spermmotiliteit, akrosoomreaksie en sperm-oesiet

binding ondersoek deur van die spesifieke PI3K inhibitor LY294002, gebruik te maak.


vi

Pl3K-inhibisie het die persentasie motiliteit en progressiewe motiliteit by

astenozoospermiese pasiënte verhoog. Onder hierdie laboratoriumtoestande het

Pl3K-inhibisie nie die akrosoomreaksie beïnvloed nie, terwyl sperm-oosiet binding

verhoog is. Hierdie resultate beteken dus dat PI3K spermmotiliteit en zona-binding

negatief beïnvloed.

In die laaste gedeelte van die studie is die effekte van intrasllulêre cGMP

akkumulasie deur akute in vivo sildenafil sitraat (ViagraTM) toediening op seminale

parameters, induksie van die akrosoomreaksie, sperm-oesiet binding en

spermmotiliteit ondersoek. Soos verwag is geen veranderinge in die makro- en

mikroskopiese seminale parameters deur sildenafil sitraat in vergelyking met plasebo

veroorsaak nie. Verder is die akrosoomreaksies ook nie deur 50mg orale sildenafil

sitraat geïnisieer of potensieer nie. Sperm-oosiet binding, geplaneerde snelheid,

reguitlyn snelheid en persentasie vinnigbewegende selle was almal vehoog na

sildenafil sitraat behandeling.

Uit hierdie resultate blyk dit dat daar verskeie rolspelers in die zona pellucida-

geïnduseerde akrosoomreaksie is. 'n Deeglike insig van al hierdie seintransduksie-

paaie en die onderlinge kruiskontak tussen mekaar sal uiteindelik die nodige inligting

rakende die reseptore waaraan hierdie seintransduksie paaie gekoppel is, verskaf

sowel as die intrasellulêre effektore wat uiteindelik spermfunksie beheer. Nog te

meer sal die begrip van hierdie regulatoriese paaie verder noodsaaklik wees vir die

toekomstige ontwikkeling van kliniese benaderings om bevrugting te bevorder of te

voorkom.


vii

This dissertation is dedicated to

Wendy and Christopher

Without your tremendous patience, support and love

I would not have been able to

successfully complete

this study.

"The art of love ... is largely the art of persistence."


viii

ACKNOWLEDGEMENTS

I wish to extend my most sincere gratitude and appreciation to the following people

for their contributions to the successful completion of this study:

Prof Daniel Franken for his guidance, encouragement, support, friendship and many

happy hours spent together in the laboratory;

Dr Carine Page for her guidance and help in the preparation of this manuscript;

My colleagues in the Department of Medical Physiology for encouraging me to

pursue the opportunity of furthering my studies;

The University of Stellenbosch for providing the research facilities;

Schering Aktiengesellschaft and the Harry Crossley Foundation for financial

support in covering research expenses;

Wendy, Christopher, my parents, family and friends for showing a keen interest in

my research and creating a support structure that enabled me to complete this study;

All those instrumental in my growth as a scientist.


ix

TABLE OF CONTENTS

Declaration

Abstract

Opsomming

Acknowledgements

List of tables

List of figures

Alphabetical list of abbreviations

Page

ii

iii

v

viii

xiv

xv

xvii

CHAPTER 1: INTRODUCTION AND STATEMENT OF PROBLEM

1.1 Introduction

1.2 Objectives and statement of the problem

1.3 Plan of study

1.4 Conclusion

1

2

2

4

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction

2.2 Capacitation

2.2.1 Cholesterol efflux and changes in membrane lipids and

phospholipids during capacitation

2.2.2 Ion fluxes and the regulation of sperm plasma membrane

potential

2.2.2.1 Modification in concentration of intracellular calcium and 17

5

6

12

15

other ions during capacitation


x

2.2.3 Changes in protein phosphorylation and protein kinase activity 23

during capacitation

2.2.3.1 Involvement of AC/cAMP/PKA pathway in capacitation 24

2.2.3.2 Involvement of PKC in capacitation 25

2.2.3.3 Involvement of tyrosine phosphorylation in capacitation 25

2.2.3.4 Crosstalk between different signalling events during 31

sperm capacitation

2.2.4 Consequences of capacitation on sperm function 33

2.3 The sperm acrosome 35

2.3.1 The acrosome reaction 36

2.3.1.1 Increase in intracellular calcium during the acrosome 43

reaction

2.3.1.2 Phospholipases activation during acrosome reaction 45

2.3.1.3 Involvement of protein kinases in acrosome reaction 46

process

2.4 Motility 49

2.4.1 Factors influencing sperm motility 52

2.4.1.1 Cyclic adenosine mono-phosphate 52

2.4.1.2 Adenylate cyclase 54

2.5 Summary 54

2.6 References 57

CHAPTER 3: MATERIALS AND METHODS

3.1 Introduction

3.2.1 Preparation of human tubal fluid culture medium

87

87


xi

3.2.2 Semen collection 88

3.2.3 Oocyte collection and storage 88

3.2.4 Solubilized zona pellucida preparation 893.2.5 Assessment of the acrosome reaction 893.2.6 Hemizona binding assay 91

3.2.6.1 Bisecting of oocytes 913.2.6.2 Competitive sperm-binding to the hemizona 92

3.2.7 Computer assisted sperm analyses 943.2.8 Statistical analyses 943.2.9 References 95

CHAPTER 4: THE ZONA PELLUCIDA-INDUCED ACROSOME REACTION

OF HUMAN SPERMATOZOA INVOLVES EXTRACELLULAR SIGNAL-

REGULATED KINASE ACTIVATION

Summary

Introduction

Materials and methods

Results

Discussion

References

CHAPTER 5: EXTRACELLULAR SIGNAL-REGULATED KINASE

ACTIVATION INVOLVED IN HUMAN SPERM-ZONA PELLUCIDA BINDING

Summary

Introduction

Materials and methods

979799102104

109

114114116


xii

Results

Discussion

References

119

121

124

CHAPTER 6: PHOSPHATIDYLINOSITOL 3-KINASE INHIBITION ENHANCES

HUMAN SPERM MOTILITY AND SPERM-ZONA PELLUCIDA BINDING

Summary 129

Introduction 129

Materials and methods 132

Results 135

Discussion 141

References 148

CHAPTER 7: THE EFFECT OF ACUTE IN VIVO SILDENAFIL CITRATE

(VIAGRA™) TREATMENT ON SEMEN PARAMETERS AND SPERM

FUNCTION

Summary 154

Introduction 154

Materials and methods 159

Results 164

Discussion 172

References 177

CHAPTER 8: SUMMARY, RECOMMENDATIONS AND CONCLUSIONS

8.1 Conclusion 182


xiii

8.1.1 Motility 1828.1.2 Acrosome reaction 184

8.2 Recommendations 1888.3 Future research 1898.4 References 191


xiv

LIST OF TABLES

Page

CHAPTER2

Table I Molecules that can induce the acrosome reaction in vivo. 40

CHAPTER 5

Table I Sperm-zona binding results after P0098059 treatment followed by 120

exposure to calcium ionophore and solubilized zona pellucida.

Table II Sperm-zona binding results expressed as a hemizona index (HZI) 121

after P0098059 (PO) treatment followed by exposure to calcium

ionophore (A23187) and solubilized zona pellucida (ZP).

CHAPTER 6

Table I Sperm kinematics results of all the samples as well as when 137

CHAPTER 7

Table I

Table II

Table III

Table IV

Table V

divided into normozoospermic and asthenozoospermic donors in

the presence and absence of LY294002.

Incidence of adverse events following treatment with placebo or 165

sildenafil citrate (n=20).

Initial macroscopic appearance and evaluation of semen after 165

either placebo or sildenafil citrate administration (n=20).

Effects of acute in vivo administration of sildenafil citrate on 166

microscopical secondary semen analysis parameters (n=20).

The effect of in vitro intracellular cGMP elevation by the addition of 167

8-Br-cGMP (20j.JM)on eliciting of the acrosome reaction (n=6).

Effects of 8-Br-cGMP (in vitro) and sildenafil (in vivo) on different

sperm motility parameters as measured by CASA.

171


xv

LIST OF FIGURES

CHAPTER2

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Page

Schematic representation of the main events occurring under 8

conditions leading to capacitation and development of

hyperactivated motility of human spermatozoa in vitro.

Working model displaying the transmembrane and intracellular 11

signalling pathways to playa role in regulating sperm capacitation.

Regulation of protein tyrosine phosphorylation by a cAMP/PKA- 30

dependent pathway.

Crosstalk between signalling pathways involved in capacitation. 32

Diagram illustrating the main signal transduction pathways 42

activated during the process of acrosome reaction in response to

zona protein 3 (ZP3).

51Figure 6 Motion parameters of a single sperm track.

CHAPTER 3

Figure 1 Patterns recorded during FITC-PSA acrosome staining. 90

Figure 2 The competitive hemizona assay. 93

CHAPTER4

Figure 1 Influence of the MEK-inhibitor PD098059 (PO) on the acrosome 103

reaction (Mean±SE) mediated by A23187.

Figure 2

Figure 3

Influence of the MEK-inhibitor PD098059 (PO) on the acrosome 104

reaction (Mean±SE) mediated by ZP.

Possible interactions between the different signal transduction

pathways invoked during the acrosome reaction.

106


xvi

CHAPTER6

Figure 1 Correlation between beat cross frequency (BCF) and progressive 138

motility (PM) of pooled experiments (n=36).

Figure 2 Correlation between beat cross frequency (BCF) and amplitude of 138

lateral head displacement (ALH) of pooled experiments (n=36).

Figure 3 Histogram showing the percentage acrosome reaction (mean±SE) 140

of control (C) spermatozoa and spermatozoa after exposure to

zona pellucida (ZP), PI3K antagonist LY294002 (LY) or both ZP

and LY294002 (ZP+LY).

Figure 4 Histogram showing the number of control and LY294002 pre- 141

treated (Test) spermatozoa (mean±SE) tightly bound to each

hemizona respectively (n=18).

Figure 5 Possible interactions between the different signal transduction 147

pathways invoked during the acrosome reaction.

CHAPTER 7

Figure 1 Enhancement of penile erection by sildenafil citrate. 158

Figure 2 The effects of double-blind placebo or 50mg-sildenafil citrate 168

administration on the sperm acrosome reaction (n=20).

Figure 3 The effect of double-blind placebo or 50mg-sildenafil citrate 169

administration on the average number of spermatozoa tightly/firmly

bound to each hemizona (n=10; P=0.281).

CHAPTER 8

Figure 1 Hypothesized signal transduction pathways and possible 187

interactions between them.


xvii

ALPHABETICAL LIST OF ABBREVIATIONS

AA

AC

AKAPs

AlHAR8-Br-cGMP

BSA

BCF

[Ca2+]1

cAMP

CASA

cGMP

DAG

OFDMSO

ED

ERKFITC-PSA

FPP

GABAA

gluNAc

G-protein

GTP

HCl

= Arachidonic acid

= Adenylate cyclase

= A kinase-anchoring proteins

= Amplitude of lateral amplitude

= Acrosome reaction

= 8-bromo-cGMP

= Bovine serum albumin

= Beat-cross frequency

= Intracellular free ionized calcium concentration

= cyclic 3' ,5' adenosine monophosphate

= Computer assisted semen analysis

= cyclic 3',5' guanosine monophosphate

= Diacylglycerol

= Decapacitation factors

= Dimethylsulfoxide/sucrose

= Erectile dysfunction

= Extracellular signal-regulated kinases

= Fluorescein-labeled Pisum Sativum agglutinin

= Fertilization-promoting peptide

= y-aminobutyric acid A

= N-acetyl-a-D-glucosamine

= Guanine nucleotide binding protein

= Guanosine triphosphate

= Hydrochloric acid


xviii

HC03- = Bicarbonate

HTF = Human tubal fluid medium

HZA = Hemizona assay

HZI = Hemizona Index

ICSI = Intracytoplasmic sperm injection

IP3 = Inositol-1, 4, 5-triphosphate

IVF = In vitro fertilization

LC = Lyso-phosphatidylcholine

LIN = Linearity

LVA = low voltage activated

MAP = Microtubule-associated proteins

MAPK = Mitogen-activated protein kinases

MEK = ERK kinase

NANC = Non-adrenergic non-cholinergic

NaOH = Sodium hydroxide

NO = Nitric oxide

P = Progesterone

PA = Phosphatic acid

PBS = Phosphate buffered saline

POEs = Phosphodiesterases

POE5 = Phosphodiesterase type 5

PI3-K = Phosphatidylinositol 3-kinase

PIP2 = Phosphatidyl-inositol biphosphate

PL = Phospholipids

PLA2 = Phospholipase A2


PlCPLO

PKA

PKC

PKG

Ptdlns

PTK

ras-MEK-MAPK

ROS

SNARE

STR

TK

TKR

VAP

VClVOCC

VOCCT

VSlWHO

ZP

ZP3

ZRK

xix

= Phospholipase C

= Phospholipase 0

= Protein kinase A

= Protein kinase C

= Protein kinase G

= Phosphatidylinositol

= Protein tyrosine kinases

= Mitogen-activated protein kinase

= Reactive oxygen species

= Soluble N-ethylmaleimide-sensitive attachment protein

receptors

= Straightness

= Tyrosine kinase(s)

= Tyrosine kinase receptor

= Average path velocity

= Curvilinear velocity (Track speed)

= Voltage-operated calcium channels

= T-type voltage-operated calcium channels

= Straight-line (progressive) velocity

= World Health Organisation

= Zona pellucida

= Zona pellucida glycoprotein 3

= Zona receptor kinase


"The great tragedy of science ...

the slaying of a beautiful hypothesis by an

ugly fact."

- Thomas Huxley-


1

CHAPTER 1

INTRODUCTION AND STATEMENT OF PROBLEM

1.1 Introduction

The development of the fertilization competent state of the spermatozoon occurs

through a series of poorly understood processes. Successful fertilization involves

several steps including (1) movement/transit from the site of ejaculation to the site of

fertilization; (2) sperm capacitation in the female genital tract; (3) binding of

capacitated spermatozoa to the oocyte's extracellular coat, the zona pellucida (ZP);

(4) induction of the acrosome reaction; (5) penetration of the ZP; and (6) fusion of the

spermatozoon with the egg vitelline membrane.

Sperm-egg interaction is a carbohydrate-mediated species-specific event that

initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal

contents (i.e. the acrosome reaction). This step is believed to be a prerequisite that

enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg.

Researchers only recently started to investigate the intracellular mechanisms

resulting in acrosomal exocytosis (i.e. fusion and vesiculation of the sperm plasma

membrane and outer acrosomal membrane, allowing the exposure and release of the

acrosomal contents). Although various studies have been published, very little work

has been done on the intracellular signalling pathways elicited by the physiologically-

induced, i.e. the zona pellucida-induced, acrosome reaction as most researchers

make use of ligands mimicking zona pellucida action, thus ultimately leading to

conflicting results.


2

1.2 Objectives and statement of the problem

Against this introductory perspective the overall objective of this research study is to

present additional data that will assist reproductive biologists and clinicians in the

ongoing quest to unravel the intracellular pathways involved in the physiologically

induced acrosome reaction. The scarcity of human zonae pellucidae has placed the

investigation into the physiological acrosome reaction out of reach of most

reproductive biologists. Due to our fortunate accessibility to human zonae pellucidae

we could subsequently persue investigations into this reaction.

The aim of this study was to investigate different members and mechanisms by which

various intracellular signalling systems are activated during the zona pellucida-

induced acrosome reaction and their specific roles in the subsequent events leading

to fertilization. Sperm-zona interaction as well as various sperm motion

characteristics was also investigated. All these studies were performed on human

spermatozoa by making use of different signal transduction inhibitors.

1.3 Plan of study

This research project includes aspects that relate to intracellular signal transduction

processes involved in human sperm function such as inducibility of the acrosome

reaction, sperm-oocyte binding and motility.

As a background to the study, a broad overview of the current literature on

capacitation, acrosome reaction and kinematics of human spermatozoa is provided in

chapter two. Following this the basic materials used and methods followed during the

research project are outlined in Chapter 3.


3

The experiments have been divided into four research papers that are presented as

separate chapters. Each article has a specific summary, introduction, material and

methods, results, discussion and reference section pertaining to the study concerned.

Although each of these chapters is a complete and separate experiment, it is

important to remember that the data remain closely related, since all the results have

a mutual goal i.e. to further our understanding of the human sperm function in the

presence of zona pellucida.

The central theme of Chapters 4 and 5 will focus on the role of extracellular signal-

regulated kinase activation in human spermatozoa during the zona pellucida-induced

acrosome reaction and its involvement in sperm-zona pellucida binding.

Chapter 6 examines the ways by which phosphatidylinositol 3-kinase inhibition

enhance sperm motility parameters, while the effects of this inhibition on the zona

pellucida-induced acrosome reaction and sperm-oocyte binding was also addressed.

The effect of sildenafil citrate on the initiation of the zona pellucida-induced acrosome

reaction, sperm-oocyte binding and motility of human spermatozoa is the subject of

Chapter 7.

The final chapter (Chapter 8) gives a retrospective look at the study. Certain aspects

of the project will be highlighted and relevant suggestions will be made.


4

1.4 Conclusion

A thorough understanding of signal transduction in human spermatozoa will

ultimately yield information regarding the nature of receptors to which these signal

transduction pathways are coupled as well as the intracellular effectors that ultimately

regulate sperm function. Moreover, an understanding of these regulatory pathways

will be essential for the future development of clinical approaches designed to

enhance or preclude fertilization.


"Live as if your were to die tomorrow.

Learn as if you were to live forever."

- Mahatma Gandhi -


5

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

In mammals testicular sperm are morphologically differentiated but are neither

progressively motile nor able to fertilize an egg. Although the ability to move forward

is acquired during epididymal maturation, sperm are fertilization incompetent until

after a finite period of residence in the female reproductive tract (Visconti et al.,

2002). Two processes, namely capacitation and acrosome reaction (AR) are of

fundamental importance in the fertilization of the oocyte by the spermatozoon.

Physiologically occurring in the female genital tract, capacitation is a complex

process, which renders the sperm cell capable for specific interaction with the oocyte.

During capacitation, modification of membrane characteristics, enzyme activity and

motility properties of spermatozoa render these cells able to penetrate oocyte

investments and responsive to stimuli that induces the AR prior to fertilization. The

physiological AR occurs upon interaction of the spermatozoon with the zona pellucida

(ZP) and specifically zona pellucida protein 3 (ZP3). This is followed by liberation of

several acrosomal enzymes and other constituents that facilitate penetration of the

zona and expose molecules on the sperm equatorial segment that allows fusion of

the sperm membrane with the oolemma (Baldi et al., 2000). The molecular

mechanisms and the signal transduction pathways mediating the processes of

capacitation and AR have been partially defined, and appear to involve modifications

of intracellular calcium and other ions, lipid transfer and phospholipid remodelling in

the sperm's plasma membrane as well as changes in protein phosphorylation. Some

of the kinases and phosphorylated proteins that are involved in the processes of


6

capacitation and AR have been characterised, while characterisation of sperm

receptors to physiological inducers of the AR is in progress. The main signal

transduction pathways involved in capacitation and AR will subsequently be

summarised, as well as the various motility parameters and factors that might

influence it.

2.2 Capacitation

The process of capacitation consists of a series of functional biochemical and

biophysical modifications that render the ejaculated spermatozoa competent for

fertilization of the oocyte. This fundamental process normally takes place in the

female genital tract during the migration of spermatozoa to the site of fertilization as a

consequence of specific interactions between sperm and epithelial tubal cells

(Yanagimachi, 1994). However, under appropriate conditions, capacitation can also

be induced in vitro (Yanagimachi, 1994). Most of our knowledge regarding this

process has in fact been derived from in vitro studies. Following ejaculation, the

sperm surface is surrounded by molecules, known as decapacitation factors (OF),

that, until released, keeps the sperm in a non-capacitated state. These factors

associate with spermatozoa following contact with seminal fluids and are

progressively released from the sperm surface during capacitation. OFs thus

modulates capacitation thereby leading spermatozoa to the maximal fertilizing ability

at the site of fertilization (Fraser, 1999). It has been supposed that OFs, once

attached to the sperm surface, activate an intracellular Ca2+ -ATPase maintaining low

intracellular calcium concentration ([Ca2+]i) (Adeoya-Osiguwa & Fraser, 1996) and

when they are released from the surface [Ca2+]i increases. Several molecules have

been indicated as possible OFs (Yanagimachi, 1994).


7

It was recently demonstrated that two proteins present in seminal plasma, uteroglobin

and transglutaminase inhibit sperm capacitation and motility, thus representing two

possible OF candidates (Luconi et aI., 2000). Another sperm inhibitory factor present

in seminal fluid is cholesterol, which is known to inhibit several sperm functions,

including capacitation (Cross, 1996; Khorasani et al., 2000). In seminal plasma there

are also molecules that can stimulate the fertilizing ability of spermatozoa such as

fertilization-promoting peptide (FPP). FPP is a small peptide that promotes

capacitation (Funahashi et aI., 2000; Fraser, 1998) and inhibits spontaneous

acrosome loss, thereby retaining fertilization potential of sperm until the site of

fertilization is reached.

Capacitation is associated with the development of a distinct motility pattern called

hyperactivation (Yanagimachi, 1994), which is characterised by pronounced flagellar

movements, marked lateral excursion of the sperm head and a non linear trajectory.

Whether the development of hyperactivated motility is related to the biochemical and

biophysical changes occurring during the process of capacitation is still a matter of

debate. It is worth noting that signal transduction mechanisms involved in the

development of this special sperm motility pattern are similar to those described to

occur during capacitation (see later). It has recently been shown that, during in vitro

capacitation, modifications of mitochondrial morphology, which may be relevant for

the development of the hyperactivated motility pattern, also occur (Vorup-Jensen et

al., 1999). An additional manifestation of sperm capacitation is the acquisition of the

ability to undergo the AR in response to physiological stimuli such as ZP3 and

progesterone (P). The responsiveness of spermatozoa to ZP3 (Florman, 1994), P


8

(Baldi et al., 1998) and other stimuli of the AR increases during capacitation, assuring

maximal responsiveness at the site of fertilization. Capacitation involves

modifications in sperm surface protein distribution, alterations in plasma membrane

characteristics, changes in enzymatic activities and modulation of expression of

intracellular constituents (Yanagimachi, 1994). These events are made possible by

activation of a cascade of signalling pathways (schematised in Figure 1) effected by

unknown mediators during transit of sperm in the female reproductive tract or during

incubation in vitro in defined media.

ROS NO

PKA ~ cAMP•PTK Tyrosine phosphorylation+)/ il??.

HYPERACTIVA TED < Capacitation

PKC

outside

o Cholesterolacceptor

Cholesterol-+t Membranefluidity membrane

inside

PLAy

Figure 1. Schematic representation of the main events occurring under conditions

leading to capacitation and development of hyperactivated motility of human


9

spermatozoa in vitro. Changes in membrane permeability to several ions have been

described, among these Ca2+ and bicarbonate (HC03-), whose influx increase during

capacitation, have been reported to exert a primary role in the process. Membrane

fluidity increases due to the loss of cholesterol from the membrane, which may be

accelerated by the presence of a cholesterol acceptor in the external medium.

Remodelling of sperm membrane phospholipids (PL) and activation of

phospholipases (PLA-2 and PLCy-1) have also been shown: in particular, increased

synthesis of phosphatidylcholine from phosphatidyl-ethanolamine,

phosphatidylinositol and Iyso-phosphatidylcholine (LC) have been documented. A

time-dependent increase of tyrosine phosphorylation of proteins is associated with

development of capacitation. The increase of tyrosine phosphorylation is primarily

dependent on the increase in bicarbonate, which, in turn, activates adenyl cyclase

(AC) with increased generation of cAMP and subsequent activation of protein kinase

A (PKA). PKA activation leads to the activation of sperm tyrosine kinase(s) (TK). On

the contrary, Ca2+ inhibits tyrosine phosphorylation during capacitation. Other

possible physiological modulators of tyrosine phosphorylation during capacitation are

reactive oxygen species (ROS) that may be generated from spermatozoa or

leucocytes present in the ejaculate and nitric oxide (NO). An involvement of protein

kinase C (PKC) and ras-MEK-MAPK (mitogen-activated protein kinase) pathways

has also been reported. (From: Baldi et ai., 2000)

UNIVERSITEIT STELLENBOSCHBIBLIOTEEK


10

All these pathways may then crosstalk among each other determining the

development of capacitation. However, the exact relationship among these

modifications is not yet completely understood. As of yet there is no well-defined

method that allows distinction of capacitated from noncapacitated spermatozoa.

Capacitation does not occur synchronously in spermatozoa (Cohen-Dayag et a/.,

1995). In addition, capacitation is transient and already capacitated spermatozoa

cannot be capacitated again (Cohen-Dayag et a/., 1995). These complexities in in

vitro capacitation make it difficult to appropriately interpret in vitro studies. To

facilitate consideration of the complex cascade of molecular events that occur during

capacitation, a discussion of this process may be divided into events that initiate

capacitation and events that are a consequence of this process. Molecular events

implicated in the initiation of capacitation include: removal of cholesterol from the

sperm plasma membrane and changes in lipid distribution and composition; ion

fluxes resulting in alteration of sperm membrane potential as well as concentration of

intracellular calcium and other ions; and changes in protein phosphorylation and

kinase activities with emphasis on an increased tyrosine phosphorylation of proteins

involved in induction of hyperactivation and the AR. A working model for the initiation

of capacitation based on recent work is presented in Figure 2.


11

Cholesterol Efflux

Hyperpolarization ofSperm Plasma

Membrane Potential

PKA Activation

Protein TyrosinePhosphorylation

Adenylate CyclaseActivation

Figure 2. Working model displaying the transmembrane and intracellular signalling

pathways hypothesised to playa role in regulating sperm capacitation. This model is

based on work from a number of laboratories (Visconti et aI., 2002).


12

2.2.1 Cholesterol efflux and changes in membrane lipids and phospholipids

during capacitation

Changes in the distribution and composition of plasma membrane lipids and PL are

an important feature of sperm capacitation. These changes lead to an increase in the

membrane fluidity and to changes in the architecture and composition of the plasma

membrane (Yanagimachi, 1994). Serum albumin, an essential component of in vitro

capacitation media, is believed to function as a sink for cholesterol by removing it

from the sperm plasma membrane (Davis et al., 1979; Davis et al., 1980; Davis,

1981; Go & Wolf 1985; Langlais & Roberts, 1985; Suzuki & Yanagimachi, 1989;

Cross, 1996; Cross, 1998). Although serum albumin may have other roles during

capacitation (Espinosa et ai., 2000), its ability to facilitate cholesterol efflux is required

for capacitation. For example, capacitation is inhibited by the addition of cholesterol

and/or cholesterol analogues to the capacitation medium (Visconti et ai., 1999b).

Furthermore, serum albumin can be substituted in in vitro capacitation media with

cholesterol-binding compounds such as high density lipoproteins (HDL) (Therien et

ai., 1997; Visconti et ai., 1999b) and cyclodextrins (Choi & Toyoda, 1998; Cross,

1999; Osheroff et al., 1999; Visconti et al., 1999a) to induce capacitation. The

removal of cholesterol and likely other sterols (e.g. desmosterol) (Visconti et ai.,

1999b) from the plasma membrane is upstream of multiple signalling events intrinsic

to the capacitation process. Visconti and co-workers (1999) have demonstrated that

heptasaccharides (cholesterol-binding molecules) promote the release of cholesterol

from the plasma membrane of mouse sperm in the absence of bovine serum albumin

(BSA), increase tyrosine phosphorylation and promote capacitation of mouse sperm

as measured by the ability of the ZP to induce the AR and by successful fertilization

in vitro. These data suggest that cholesterol release is the signal that activates


13

membrane signal transduction pathways related to capacitation (Visconti et al.,

1999).

The removal of sterols decreases the cholesterol:phospholipid molar ratio in the

sperm plasma membrane as assessed by different criteria (Davis, 1981; Tesarik &

Flechon, 1986; Hoshi et al., 1990; Cross 1998). This could account for the membrane

fluidity changes (Wolf & Cardullo, 1991; Wolf et al., 1986) and redistribution of

membrane proteins, observed with lectins (Cross & Overstreet, 1987) and antibodies

(Shalgi et al., 1990; Rochwerger & Cuasnicu, 1992) that occur during capacitation.

From the standpoint of cell signalling, capacitation-associated alterations, in the

transbilayer phospholipid behaviour resulting in membrane lipid disorders, were

recently reported to occur through a cAMP-dependent pathway after exposure of

boar sperm to HC03- (GadelIa & Harrison, 2000). Therefore, multiple plasma

membrane modifications appear to contribute to the process of capacitation. It is also

important to consider what component of the female tract fluid might serve as a

cholesterol acceptor in vivo. Since fluids of the female tract are partially derived from

serum, serum-associated sterol acceptors could function in vivo. The identity of such

acceptors remains to be clarified.

The total amount of PL does not appear to change considerably during capacitation

(Yanagimachi, 1994). However, capacitation is associated with an increase of

phospholipid methylation and increased synthesis of phosphatidylcholine from

phosphatidylethanolamine (Llanos & Meizei, 1983). Incubation of spermatozoa under

capacitating conditions, in the presence of bicarbonate, does not alter phospholipid

distribution (Harrison & GadelIa, 1995). Such conditions, however, strongly inhibits


14

phospholipid transfer and leads to a slow increase of phosphatidylcholine

concentration in the inner leaflet of the membrane (Harrison & GadelIa, 1995).

Recently, GadelIa and Harrison (2000) have shown that the inclusion of bicarbonate

in the capacitating medium increases the translocation of phosphatidylcholine and

sphingomyelin from the outer to the inner leaflet of the membrane, possibly due to

activation of a bi-directional translocase (scramblase). Levels of phosphatidylinositol

and LC increase during capacitation in vivo in porcine sperm (Snyder & Clegg, 1975).

In view of the fusogenic properties of Iysophospholipids, an increase in their relative

amount may be relevant to prepare the sperm for the AR.

The molecular basis of signalling events induced by cholesterol efflux from sperm is

not well understood. In somatic cells, cholesterol removal is thought to disrupt lipid

rafts activating signalling events involving tyrosine kinases (TK), guanine nucleotide

binding proteins (G-proteins), and/or other molecules (Kabouridis et aI., 1997; Brown

& London, 1998; Roy et aI., 1999). The activation of similar signalling events during

capacitation in sperm correlates to the removal of cholesterol from the plasma

membrane. In sperm, cholesterol may likewise be concentrated in specialised plasma

membrane microdomains such as lipid rafts and caveolae (Brown & London, 1998).

This idea is supported by the recent finding that one important component of

caveolae, caveolin 1, is present in the plasma membrane overlying the acrosomal

region and the flagellum of mouse and guinea pig sperm (Travis et aI., 2000). The

hypothesis that lipid rafts and caveolae concentrate signalling complexes in the

sperm plasma membrane warrants continued investigation.


15

2.2.2 Ion fluxes and the regulation of sperm plasma membrane potential

During transit through the male and female reproductive tracts, sperm are exposed to

significant changes in the extracellular milieu, including variations in extracellular ion

concentrations and osmolarity. For example, caudal epididymal sperm are stored in

an environment that contains high K+, low Na+ and very low HC03- concentrations

(Brooks, 1983; Setchell et a/., 1994). These ion concentrations radically change when

the sperm are ejaculated, first in the seminal fluid and then in the female tract, where

the K+ is significantly reduced and the Na+ and HC03- concentrations are significantly

increased (Brooks, 1983; Setchell et a/., 1994; Yanagimachi, 1994). These dramatic

shifts in extracellular ion concentrations trigger modulations in intracellular ion

concentrations and consequently lead to an altered membrane potential of the sperm

plasma membrane.

Changes in intracellular ion concentrations have been associated with different

aspects of sperm function such as sperm motility in trout sperm (Morisawa & Suzuki,

1980; Gatti et a/., 1990), capacitation in mammalian sperm (Visconti et a/., 1995a;

Zeng et a/., 1995; Arnouit et a/., 1999) and the acrosome reaction in sperm from

multiple species (Arnouit et a/., 1996; Darszon et a/., 1999). The dramatic influence of

the external ion composition and the effect of channel blockers on sperm motility,

capacitation, and the AR strongly suggest that ion channels actively participate in the

regulation of sperm function. Ion channels can catalyse the flow of millions of ions

through the non-conducting lipid bilayer; therefore, a few ion channels can cause

changes in a small cell like the sperm within milliseconds (Darszon et aI., 1999). Ion

concentrations determine the plasma membrane potential through ion-selective

channels and control the extent of channel activity and ion flow. The plasma


16

membrane potential can also regulate ion channel activity as well as second

messenger levels. For example, in trout sperm, changes in the plasma membrane

potential by changes in extracellular K+ concentration modulate sperm motility

through a cAMP pathway (Morisawa & Ishida, 1987). Moreover, Beltran et ai., (1996),

demonstrated in sea urchin sperm that cAMP synthesis could be regulated by

changes in membrane potential.

In vitro, the resting membrane potential is determined by the relative permeabilities of

the sperm plasma membrane for ions that constitute the capacitation media. Under

normal conditions, sperm maintain an internal ion concentration markedly different

from that in the extracellular medium and these differences establish the resting

plasma membrane potential. The ion composition of capacitation media mimics that

of oviductal fluid (Yanagimachi, 1994). These media are high in Na+ (about 130 mM)

and cr (about 100 mM), but low in K+ (about 5.9 mM). Capacitation media also

contain Ca2+ (about 2.7 mM) and HC03- (10-25 mM). In contrast, intracellular fluids of

sperm have a low concentration of Na+ (about 14 mM) and high concentration of K+

(about 90-120 mM) (Babcock, 1983; Zeng et ai., 1995). The free intracellular Ca2+

concentration of non-capacitated sperm is approximately 0.1 M or less, but during the

AR it may increase to approximately 10M (Bailey & Storey, 1994; Arnouit et al.,

1999). To date, the intracellular concentrations of cr and HC03- in sperm have not

been determined. These differences between extracellular and intracellular ion

concentrations are established by the respective ion permeabilities and determine the

resting membrane potential in mammalian sperm.


17

Recently, Zeng et a/., (1995) demonstrated that capacitation is accompanied by

hyperpolarization of the sperm plasma membrane. Membrane hyperpolarization may

be partially due to an enhanced K+ permeability related to the decrease in inhibitory

modulation of K+channels during capacitation (Zeng et a/., 1995). Since capacitation

prepares the sperm for the AR, capacitation-associated hyperpolarization may

regulate the ability of sperm to generate transient Ca2+ elevations during AR by

physiological agonists (e.g. ZP of the egg or P). This hypothesis is consistent with the

presence of low voltage activated (LVA) Ca2+ T-channels in spermatogenic cells

(Arnouit et a/., 1996; Lievano et a/., 1996) that may also be present in mature sperm.

A signature property of LVA Ca2+ channels is a low threshold for voltage-dependent

inactivation. These Ca2+ channels are inactivated at holding potentials between -80

and -60 mV and cannot be activated readily from more positive holding potentials

(Arnouit et a/., 1996; Lievano et al., 1996). Thus, if LVA Ca2+ T-channels are involved

in the regulation of the AR, sperm must maintain a hyperpolarized membrane

potential during the early stages of interaction with the egg (Arnouit et a/., 1999;

Florman et al., 1998). Presently, little is known about the regulation of capacitation-

associated hyperpolarization. In order to understand the ionic basis of these changes

in sperm plasma membrane potential, it will be necessary to analyse how the

aforementioned ion permeabilities change during capacitation.

2.2.2.1 Modification in concentration of intracellular calcium and other ions

during capacitation (HC03-, Ca2+, and the cAMP pathway)

Numerous studies have demonstrated that capacitation is Ca2+ -dependent

(DasGupta et al., 1993; Visconti et a/., 1995a). The initiation and/or regulation of

capacitation by Ca2+ occur via different targets, some of which are involved with


18

cAMP metabolism. Since in sperm Ca2+/calmodulin can activate both the synthesis of

cAMP by adenylate cyclase (Gross et aI., 1987), as well as degradation by cAMP

cyclic nucleotide phosphodiesterase (Wasco & Orr, 1984), it is not surprising that

Ca2+ has both positive and negative actions on capacitation and related signalling

events. In this respect, Ca2+ has a positive effect on mouse sperm by inducing

capacitation-associated changes in protein tyrosine phosphorylation (Visconti et al.,

1995a). In contrast, Ca2+ has been demonstrated to inhibit protein tyrosine

phosphorylation in human sperm during the first 2 h of in vitro capacitation (Carrera

et aI., 1996; Luconi et aI., 1996). An increase in intracellular sperm Ca2+ during

capacitation has been described by some investigators, whereas others have shown

that no changes in Ca2+ levels occur during this maturational event (Yanagimachi,

1994). This ambiguity could be due, in part, to the well-demonstrated action of Ca2+

on the AR and to the inherent difficulties in differentiating between these events.

However, the action of Ca2+ at the level of effector enzymes involved in sperm signal

transduction suggests that this divalent cation is likely to play an important role in

capacitation.

Modification of intracellular concentration of calcium ions ([Ca2+]i) is the most fully

characterised biochemical event during capacitation. An increase in the concentration

of Ca2+ during capacitation has been demonstrated in several mammalian species

(Yanagimachi, 1994) including human (Baldi et aI., 1991; Garcia & Meizei, 1999).

Extracellular Ca2+ is indeed one of the necessary constituents for the completion of

capacitation of spermatozoa in vitro (Yanagimachi, 1994). In spermatozoa [Ca2+]i is

regulated by a Ca2+-ATPase (acting as a Ca2+ extrusion pump) (Fraser & McDermott,

1992), Ca2+/H+ exchanger system and Na+/Ca2+ antiporter (acting as Ca2+ entrance


19

systems) in the plasma membrane (Fraser, 1995), and by putative intracellular Ca2+

stores, whose presence in human sperm has been suggested by several evidences

(Blackmore, 1992; Walensky & Sneider, 1995; Dragileva et ai., 1999; O'Toole et ai.,

2000). However, the role of intracellular calcium stores in the physiology of

spermatozoa is still questioned (O'Toole et ai., 2000; Kirkman-Brown et ai., 2000;

Kobori et al., 2000). Recent data (Dragileva et al., 1999) indicate that cytosolic Ca2+

is actively transported into the acrosome by an ATP-dependent, thapsigargin-

sensitive pump and that it may be released from the acrosome through an inositol-

1,4,5-triphosphate (IP3)-gated calcium channel. More recently, the existence of store-

operated calcium channels that mediate sustained calcium increase in response to

ZP3 in mouse sperm has been demonstrated (O'Toole et ai., 2000). The location of

such stores remains to be demonstrated since endoplasmic reticulum is not present

in mature spermatozoa and the acrosome does not appear (according to recent

studies performed in individual spermatozoa) to retain significant amounts of calcium

(Kirkman-Brown et ai., 2000; Kobori et ai., 2000). It has been hypothesised that

modulation of the activity of the Ca2+-extrusion system, in particular Ca2+-ATPase,

occurs during capacitation leading to an increase in intracellular Ca2+ (Fraser &

McDermott, 1992; Fraser, 1995). Drugs such as quercetin, that inhibit Ca2+-ATPase,

accelerate capacitation (Fraser & McDermott, 1992; Fraser, 1995). A Na+/Ca2+

exchanger is present in mammalian sperm, however, its role in controlling

intracellular Ca2+ during capacitation is not clear. Similarly, although voltage-operated

calcium channels (VOCG) have been demonstrated in mammalian spermatozoa

(Benoff, 1998), their role in regulating intracellular calcium concentration during

capacitation is not clear. Moreover, although the presence of T-type voltage-operated

calcium channels (VOCCT) in germ cells has been unequivocally demonstrated


20

(Arnouit et al., 1996; Darszon et al., 1999) and their involvement in the effect of ZP3

on mature sperm indicated (Arnouit et ai., 1996, O'Toole et ai., 2000), the role of

these channels in the process of capacitation is far from being defined. However, it

seems quite clear that sperm VOCCT may be modulated during capacitation. Indeed,

during this process the sperm membrane becomes hyperpolarized due to enhanced

K+ permeability (Brewis et al., 2000). This hyperpolarization may act to prime VOCCT

from an inactivated state to a closed one, which can be activated by an agonist

inducing depolarisation such as ZP3 (Arnouit et ai., 1998). Downstream targets of

calcium include the calcium-binding protein calmodulin, whose involvement in sperm

capacitation has been recently demonstrated (Si & Olds-Clarke, 2000). Besides Ca2+,

intracellular K+ (Zeng et ai., 1995), Na+ (Hyne et ai., 1985) and cr (Fraser, 1995)

concentrations have been shown to be modulated during capacitation. The increase

of intracellular Na+ appears to be important for capacitation, since the Na+ ionophore,

monensin, promotes this process in mouse sperm (Fraser, 1995). The intracellular

concentration in zinc ion decreases in the acrosome of hamster spermatozoa during

capacitation (Andrews et ai., 1994). In addition, incubation of spermatozoa in a zinc-

containing medium inhibits the process (Andrews et ai., 1994). These findings

suggest that zinc may play a role in destabilisation of plasma membrane during

capacitation (Andrews et ai., 1994).

A rise in intracellular pH has been reported during capacitation of bovine sperm

(Vredenburgh-Wilberg and Parrish, 1995). However, the role of pH in sperm

capacitation is not yet clear, since an artificial increase in intracellular pH in

spermatozoa does not accelerate the process (Fraser, 1995). Cross et al. (1997)

have shown that a cholesterol efflux from the plasma membrane, during the


21

capacitation of human spermatozoa, determines a rise in intracellular pH and

responsiveness to P. Recent data have demonstrated the presence of a Na+

dependent Cr/HC03- exchanger that regulates bicarbonate transport and motility in

mature sperm (Zeng et al., 1996), however the role of this pump in capacitation is not

clear. It has been shown that bicarbonate regulates adenylate cyclase (AC) activity

and cAMP metabolism (Visconti et al., 1990) and is necessary for tyrosine

phosphorylation of proteins during capacitation (see later). It is of interest that

bicarbonate concentration is low in the epididymis and high in the seminal plasma

and in the oviduct indicating that modifications of bicarbonate concentration in

reproductive tracts play an important role in the suppression of capacitation in the

epididymis and in the promotion of this process in the female reproductive tract

(Wassarman, 1999).

Numerous studies have demonstrated that capacitation is HC03--dependent (Lee &

Storey, 1986; Neill & Olds-Clarke, 1987; Boatman & Robbins, 1991; Shi & Roldan,

1995; Visconti et ai., 1995a). Little is known about the mechanisms of HC03-

transport in sperm. However, the ability of 4,4'-diidothiocyanatostilbene-2,2'-disulfonic

acid (OIOS), a well-known inhibitor of anion transporters, to inhibit the actions of

HC03- on various sperm functions suggests that sperm contain functional anion

transporters (Okamura et al., 1988; Visconti et al., 1990; Spira & Breitbart,

1992;Visconti et ai., 1999c).

The transmembrane movement of HC03- anions into sperm could be responsible for

the known increase in intracellular pH that is observed during capacitation (Uguz et

al., 1994; Zeng et al., 1996). An additional target for the action of this anion could be


22

the regulation of sperm cAMP metabolism, since the synthesis of cAMP by

mammalian sperm AC is markedly stimulated by HC03- (Okamura et aI., 1985; Garty

& Salomon, 1987; Visconti et aI., 1990). The increase in cAMP during capacitation

and the stimulation of AC activity in sperm by increased levels of intracellular HC03-

and Ca2+ implicate a role for this enzyme and the cAMP-signalling pathway in

capacitation. AC in sperm has been the subject of multiple studies, but whether one

or more proteins represent it remains controversial.

Sperm AC has unique properties when compared with somatic cell cyclases (Garbers

& Kopf, 1980; Leclerc & Kopf, 1995). For example, unlike somatic cell cyclases,

responses of sperm AC to agents that modulate stimulatory GTP binding proteins

(Gs), such as cholera toxin, AIF4- or GTP analogues, are weak or completely absent.

Since no cholera toxin-ADP ribosylated substrate has been detected in mammalian

sperm, the low sensitivity to G-protein effectors could be due to the lack of Gs protein

in these cells (Hildebrandt et aI., 1985). Another possibility is that the sperm AC is

unable to interact with Gs proteins due to differences in cyclase tertiary structure. As

mentioned above, an important property of the sperm AC is its regulation by HC03-

anion (Okamura et al., 1985).

Recent studies suggest that the sperm AC is a post-translationally modified form of

the testicular soluble AC (Buck et al., 1999). Similar to the sperm AC activity, the

enzymatic activity of recombinant testicular soluble AC is stimulated by HC03- anions

(Chen et al., 2000). In addition, antibodies against the catalytic domain of the

testicular soluble AC recognised two sperm proteins in corresponding to the deduced

molecular masses of the processed and unprocessed forms of the testicular enzyme


23

(Chen et al., 2000) suggesting that this cyclase remains associated with sperm after

spermatogenesis. Interestingly, the sequence from the catalytic domain of this

cyclase has sequence homology to cyanobacterial AC and the cyanobacterial

cyclase is also HC03--dependent (Chen et aI., 2000). Although the testis cyclase has

been found in the soluble fraction, it is significant that cyclase activity identified in

mammalian sperm remains associated with the particulate membrane fraction.

Therefore, the testicular soluble AC found would be predicted to have a mechanism

allowing for translocation from the cytosol to the membrane at some point during

spermatogenesis.

2.2.3 Changes in protein phosphorylation and protein kinase activity during

capacitation

Protein phosphorylation during sperm capacitation has been widely studied in the last

five years, leading to the generation of more than 100 papers in the international

literature. The best studied kinases involved in capacitation are Ca2+-calmodulin

activated kinases, cAMP-dependent kinases (pKA), calcium and phospholipid

activated protein kinase (PKC), which induce phosphorylation of proteins in serine

and threonine residues, and TKs, which phosphorylate proteins in tyrosine residues.

The involvement of kinase pathways in the process of capacitation has been mainly

studied by using inhibitors of such pathways. However, specificity of kinase inhibitors

is doubtful and many of them may affect other intracellular signal transduction

pathways. In this light, caution should be applied in interpreting results of these

studies (Baldi et al., 2000).


24

2.2.3.1 Involvement of AC/cAMP/PKA pathway in capacitation

A spontaneous increase of cAMP during capacitation has been demonstrated (White

& Aitken, 1989) and inhibitors of PKA, the serine-threonine kinase activated by this

pathway, inhibit capacitation (Aitken et ai., 1998). Pentoxifylline, which promotes an

increase in cAMP by inhibiting sperm phosphodiesterases, induces capacitation (Ain

et ai., 1999). It has been well established that Ca2+ and HC03- stimulate AC but the

exact mechanism by which these ions activate AC is not clear (Visconti et al., 1990;

Garty & Salomon, 1987). cAMP generated during capacitation and subsequent

activation of PKA appears to play a key role in the increase of tyrosine

phosphorylation during capacitation (Visconti et ai., 1999, also see later). Since PKA

may phosphorylate several cellular substrates, its sequestration in specific cellular

compartments is necessary to spatially restrict its action, thus ensuring specificity of

functions. Compartmentalisation of PKA is accomplished by A-kinase-anchoring

proteins (AKAPs) which have been recently characterised in sperm of different

species (Carerra et al., 1996; Moss et ai., 1999; Vijayaraghavan et ai., 1999). Their

specific localisation to the tail of the sperm indicates a role for these proteins in the

modulation of sperm motility (Vijayaraghavan et ai., 1999). Recently, a role for the

AC/cAMP/PKA pathway in plasma membrane lipid remodelling, occurring during

capacitation, has been demonstrated (Harrison & Miller, 2000). Yet, many questions

still remain unanswered concerning the AC/cAMP/PKA pathway in human sperm.

Although the specific expression of a soluble AC has been shown in male germ cells

(Sinclair et ai., 2000), the types of phosphodiesterases (PDEs) present in these cells

remain to be defined. Recently, Richter et al. (1999) detected, the presence of mRNA

transcripts for several PDEs in ejaculated human spermatozoa, but no conclusions


25

can be drawn concerning the type of POE specifically expressed in mature

spermatozoa.

2.2.3.2 Involvement of PKC in capacitation

The presence of PKC in mammalian spermatozoa and its role in sperm motility and

the process of AR are documented (Breitbart & Noar, 1999), but PKC activity is very

low compared to somatic cells (Breitbart & Noar, 1999, Bonaccorsi et a/., 1998). The

role of this enzyme during capacitation is poorly understood. Early studies

demonstrated that stimulation of PKC with phorbol esters accelerates the process of

capacitation (Rotem et a/., 1992). This effect was inhibited by PKC inhibitors,

suggesting that PKC may be involved in capacitation (Rotem et a/., 1992). In

addition, PKC may also be involved in epidermal growth factor-induced capacitation

(Furuya et a/., 1993). All these studies were performed using high levels of phorbol

esters as PKC inducers. Although it was shown that PKC activity of human sperm

can be stimulated by a phorbol ester (Bonaccorsi et a/., 1998), additional effects of

these tumour promoters on other sperm kinases cannot be excluded. Recently, the

role of PKC in capacitation has been questioned (Ain et a/., 1999).

2.2.3.3 Involvement of tyrosine phosphorylation in capacitation

The first evidence for the presence of tyrosine phosphorylated proteins in mammalian

spermatozoa dates back to 1989 (Leyton & Saling, 1989). Using anti-

phosphotyrosine antibodies, Leyton and Saling (1989) identified three different

phosphoproteins at 52, 75, and 95 kOa in the mouse spermatozoa. The 95 kOa

protein was tyrosine phosphorylated under all experimental conditions and including

interaction of spermatozoa with solubilized ZP proteins (Leyton & Sailing, 1989).


26

Capacitation is characterised by a spontaneous, time-dependent increase of tyrosine

phosphorylation of different proteins (Luconi et al., 1995; Visconti et al., 1995). The

main tyrosine phosphorylated proteins are in the range of 95-100 kDa (Leyton &

Saling, 1989; Luconi et al., 1995; Visconti et al., 1995; Luconi et al., 1996; Osheroff

et al., 1999). Capacitation-associated changes in protein tyrosine phosphorylation

have been demonstrated in multiple species including the mouse (Visconti et al.,

1995a), bovine (Galantino-Homer et al., 1997), human (Leclerc & Kopf, 1995;

Osheroff et ai., 1999), pig (Kalab et ai., 1998) and hamster (Devi et ai., 1999; Visconti

et al. 1999c; Jha & Sjivaji, 2001). In the mouse, in vitro capacitation of caudal

epididymal sperm promotes tyrosine phosphorylation of a subset of proteins between

Mr 40000 and 120000 (Visconti et al., 1995a). At least in the mouse, the increase of

protein tyrosine phosphorylation is dependent on the presence of Ca2+, NaHC03- and

BSA (Visconti et ai., 1995). Specifically, the absence of anyone of these media

constituents prevents both protein tyrosine phosphorylation and capacitation. It is

necessary to mention that the effect of media constituents on protein tyrosine

phosphorylation and capacitation varies slightly from species to species (Visconti et

ai., 1999c; Jha & Sjivaji, 2001). It is worth to note that some of these results have not

been confirmed in human sperm. Indeed, in humans, Ca2+ and tyrosine

phosphorylation seem to be inversely related and protein tyrosine phosphorylation is

enhanced in calcium free medium (Luconi et al., 1996; Carrera et al., 1996) indicating

that calcium might induce the activation of tyrosine phosphatases (Carrera et al.,

1996). The dependence of in vitro protein tyrosine phosphorylation on serum

albumin, or other cholesterol acceptors, indicates a correlation between cholesterol

efflux and cAMP-induced tyrosine phosphorylation. It has been demonstrated that in

BSA-deprived media protein tyrosine phosphorylation and sperm capacitation are


27

inhibited (Visconti et al., 1995; Osheroff, 1999). Whether cholesterol removal is

upstream from or co-incidental with the action of Ca2+ and/or NaHC03" is not

presently known. It is hypothesised that cholesterol removal, with a resultant change

in sperm plasma membrane fluidity, modulates Ca2+ and/or HC03" ion fluxes leading

to AC activation; this hypothesis remains to be tested.

The increase in protein tyrosine phosphorylation is regulated by an AC/cAMP-

dependent pathway that involves PKA in sperm from the mouse (Visconti et ai.,

1995b), bull (Galanti no-Homer et ai., 1997), human (Leclerc et ai., 1996; Osheroff et

ai., 1999), boar (Kalab et ai., 1998) and hamster (Visconti et ai., 1999c), as well as by

reactive oxygen species generated at the beginning of capacitation (LeClerc et al.,

1998; see later). In addition, recent data suggest a role for nitric oxide generated by

human spermatozoa in the promotion of capacitation (Herrero et al., 1998;

Francavilla et al., 2000) and the increase of tyrosine phosphorylation (Herrero et al.,

1998).

The involvement of PKA is indicated since inhibitors of PKA activity are able to inhibit

tyrosine phosphorylation as well as capacitation. Since PKA is not able to

phosphorylate proteins on tyrosine residues, an intermediate TK may be involved.

Figure 3 summarises three possible mechanisms: (1) direct or indirect stimulation of

a TK by PKA; (2) direct or indirect inhibition of a phosphotyrosine phosphatase; and

(3) direct or indirect phosphorylation of proteins by PKA on serine or threonine

residues that prime these proteins for subsequent phosphorylation of tyrosine

residues. These distinct possibilities are currently being explored in different


28

laboratories. The identification of the enzymes responsible for the tyrosine

phosphorylation pathway(s) will improve our knowledge of the capacitation process.

As stated above, in human sperm the highest degree of tyrosine phosphorylation was

found in a protein of 95-97 kDa (Leyton & Saling, 1989; Luconi ef al., 1995; Visconti

ef al., 1995; Luconi ef al., 1996; Osheroff, 1999). A tyrosine-phosphorylated protein in

this molecular weight range was previously indicated as the possible sperm receptor

for ZP3, zona receptor kinase (ZRK) (Burks ef al., 1995). This protein has been

characterised, partially cloned and sequenced. Its sequence shows a 55% homology

with the receptor-like PTK c-eyk (Burks ef aI., 1995) and 97-100% homology with the

proto-oncogene c-mer (Bork ef al., 1996). Among the other tyrosine phosphorylated

proteins in the same molecular weight range, an AKAP specifically expressed at the

tail level has been recently identified (Carrera ef al., 1996; Moss ef al., 1999;

Vijayaraghavan ef al., 1999). This protein may be involved in the development of the

hyperactivated motility pattern. An extracellular signal regulated kinase pair (ERK-1

and ERK-2) of sperm proteins that are phosphorylated on tyrosine and activated

during sperm capacitation was also identified (Luconi ef al., 1998; Luconi ef al.,

1998). Their inhibition with a pharmacological compound suppresses capacitation

(Luconi ef al., 1998), indicating a role for these proteins in the process.

Immunofluorescence labelling of phosphotyrosine residues, indicated that

capacitation as well as exposure to zona proteins increased the degree of tyrosine

phosphorylation in each spermatozoon and the number of sperm cells

phosphorylated in the acrosomal region of the sperm head (Naz ef aI., 1991).

Incubation of spermatozoa with antiphosphotyrosine antibodies or inhibition of TK

activity inhibited zona-free hamster egg penetration (Naz ef al., 1991), prevented AR


29

and blocked fertilization (Leyton et ai., 1992). However, whether tyrosine

phosphorylation is fundamental for the development of the capacitated state or is

simply associated to the phenomenon stay a matter of debate. It was also shown that

erbstatin, a potent inhibitor of TK, did not inhibit capacitation measured as the ability

of spermatozoa to respond to P (Luconi et ai., 1996). Similarly, Ain et al. (1999), have

reported that thyrphostin A-47, a PTK inhibitor, does not inhibit pentoxifylline-

stimulated capacitation, although it suppress the AR stimulated by this agent. To

understand the role of tyrosine phosphorylation in the process of capacitation, we will

probably need to wait for the characterisation of many, if not all, of the tyrosine

phosphorylated proteins as well as the TKs that are activated during the process

(Baldi et al., 2000).


30

Protein Kinase A

t Tyrosine Kinase SerlThrPhosphorylation

I Phosphotyrosine'" Phosphatase

TyrosinePhosphorylation

Other kinases

Figure. 3. Regulation of protein tyrosine phosphorylation by a cA,MPIPKA-dependentltp

pathway. (1) A protein tyrosine kinase is stimulated through direct phosphorylation by

PKA or by an enzymatic cascade that involves phosphorylation by PKA. (2) A

phosphotyrosine phosphatase is inhibited through direct phosphorylation by PKA or

by an enzymatic cascade that involves phosphorylation by PKA. (3) Protein targets

become substrates for protein tyrosine activity after phosphorylation in ser or thr

residues by PKA (Visconti et aI., 2002).


31

2.2.3.4 Crosstalk between different signalling events during sperm capacitation

Cyclic AMP appears to be a central regulator of several sperm processes, such as

motility (Eddy & O'Brien, 1994), capacitation (Visconti et al., 1995b; Visconti et al.,

1997), and the AR (De Jonge et al., 1991a; Leclerc & Kopf, 1995; Garde & Roldan,

2000). Changes in membrane potential are also involved in these sperm functions

(Morisawa & Suzuki, 1980; Florman et al., 1992; Zeng et al., 1995; Arnouit et al.,

1996; Darszon et al., 1999). Presently, it is not known whether these two

(capacitation and AR) signalling events interact. However, it is likely that these two

processes are related, since capacitation is accompanied by both hyperpolarization

of the plasma membrane and an increase in cAMP synthesis. Supporting this idea,

the presence of a membrane potential-regulated AC has been reported in non-

mammalian species (Beltran et aI., 1996). In addition, although PKA is the main

downstream effector for cAMP in sperm, the view that PKA mediates all of the effects

of cAMP has been amended with the discovery of new types of cyclic nucleotide

receptors. These receptors include cyclic-nucleotide-gated channels, exchange

factors (Kawasaki et al., 1998), a cGMP binding cyclic nucleotide POE, and

extracellular cAMP receptors (Shabb & Corbin, 1992). Cyclic nucleotide-gated

channels were identified in sea urchin (Gauss et al., 1998) and mammalian sperm

(Weyand et al., 1994) with specificity for cAMP and cGMP, respectively. Altogether,

these data support the idea that crosstalk might occur between modification in

membrane potential and the cAMP signalling pathway during capacitation. Alternative

possibilities are summarised in Figure 4.


32:--------------------------------------------: (3), ,

eSA --'1 eSA-chol. ,(a)

- ~ I ~b)..-fr- L__ C_h_O_I.__ Hyperpolarization ~ .-} tAC -tcAMP - tPKA

1 (2) tHCO;

*/+tHC03-

tAC --. tcAMP --. tPKA --. Hyperpolarization

CAMP-gated channel 1

Figure 4. Crosstalk between signalling pathways involved in capacitation. (1)

Hyperpolarization is upstream to the increase in cAMP synthesis. Cholesterol

removal regulates sperm plasma membrane potential through a I<" channel or

through the increase in anionic permeability, (a) hyperpolarization may regulate AC

activity as described in sea urchin and trout sperm (Beltran et aI., 1996; Morisawa &

Ishida, 1987) or (b) hyperpolarization may regulate HC03- permeability and in this

way activate AC. (2) Hyperpolarization is downstream of the increase in cAMP

synthesis. In this model, cholesterol removal regulates an HHC03- permeability and

HC03- stimulates AC, cAMP could then activate a cyclic nucleotide gated channel

directly or indirectly through phosphorylation by PKA leading to plasma membrane

hyperpolarization. (3) Hyperpolarization and cAMP synthesis are independently

associated with capacitation.


33

2.2.3.5 Consequences of capacitation on sperm function

Although fertilization still represents the endpoint of sperm capacitation, the ability of

the sperm to undergo a regulated AR (e.g. in response to the ZP or P) can be taken

as an earlier, upstream endpoint of capacitation (Florman & Babcock, 1991; Visconti

et al., 1998). Capacitation is also correlated with changes in sperm motility patterns,

designated as hyperactivation, in a number of species (Suarez, 1996; Yanagimachi,

1994). When one attempts to understand the process of capacitation at the molecular

level, events occurring both in the sperm head (i.e. AR) and in the tail (i.e. motility

changes) must be considered. Therefore, one may postulate that components of both

the sperm exocytotic and motility machinery are modified during capacitation. Some

of these alterations may involve changes in the phosphorylation status of certain

proteins, changes in protein localisation, and/or modification of protein-protein

interactions. Experiments leading to the identification and characterisation of these

effector molecules will further increase our understanding of capacitation.

To understand the link between capacitation and the AR, a better knowledge of the

mechanisms that regulate this exocytotic event in sperm is necessary. Exocytosis is

a tightly regulated, complex process that involves fusion of subcellular vesicles with

the overlying plasma membrane and release of vesicular contents. Recent evidence

suggests that membrane fusion is governed by a few conserved protein families

regardless of whether membrane fusion occurs between intracellular organelles or

between trafficking vesicles and the plasma membrane. Proteins involved in fusion

events include a family of proteins commonly referred to as SNARE proteins (soluble

N-ethylmaleimide-sensitive attachment protein receptors) (Jahn & Sudhof, 1999).


34

Sperm homologues of SNARE proteins as well as SNARE-associated proteins, such

as Rab 3A and NSF, have been detected in sea urchin (Schulz et ai., 1997; Schulz et

al., 1998) and mammalian sperm (Michaut et al., 2000; Ramalho-Santos et al., 2000;

Yunes et al., 2000). These observations support the idea that the sperm AR might be

regulated in similar ways to exocytotic processes in somatic cells. Since capacitation

is necessary for exocytosis in mammalian sperm, elucidation of the mechanisms

regulating the AR will also increase our understanding of capacitation.

Capacitation is also linked to events that occur in the sperm flagellum. For example,

two members of the AKAP family located in the fibrous sheath become

phosphorylated at tyrosine residues during human sperm capacitation (Carrera et al.,

1996; Mandai et ai., 1999; Vijayaraghavan et ai., 1999). AKAPs represent a growing

family of scaffolding proteins that function to tether the regulatory subunits of PKA

and signalling enzymes, such as calcineurin and PKC, to organelles or cytoskeletal

elements. These proteins permit the precise control of signal transduction in discrete

regions of the cell (Pawson & Scott, 1997). Tyrosine phosphorylation of AKAPs might

alter the biochemical and biophysical properties of these proteins and the fibrous

sheath and thus, contribute to the regulation of events associated with flagellar

bending, including changes in tail wave amplitude during hyperactivation.


35

2.3 The sperm acrosome

The acrosome plays an important role at the site of sperm-zona (egg) binding during

the fertilizing process. Clinical studies have identified a distinct group of men whose

infertility is associated with abnormal AR (Benoff, 1997). Since the acrosome acts in

concert with the plasma membrane overlying the acrosome during the early events of

fertilization, any discussion on its formation and organisation will contribute to our

understanding of its functional significance.

The acrosome is a Golgi-derived secretory granule that is formed during an early

stage of spermiogenesis. It resembles the cellular lysosome, a bag like structure that

normally functions in intracellular digestive and defensive mechanisms, in three

different ways. First, both the acrosome and the lysosome are derived from the Golgi

apparatus. Secondly, both organelles stain a bright orange red colour with acridine

orange, indicating an acidic pH within the organelles. Finally the two organelles

contain several common enzymes such as acid glycohydrolases, proteases,

esterases, acid phosphatases and aryl sulfatases (Tuisiani et al., 1998; Zaneveld &

De Jonge, 1991). Despite these similarities the acrosome has some distinctive

features. The sperm acrosome is a sac-like structure surrounded by inner and outer

acrosomal membranes. Immediately after sperm (receptor)-zona ligand) binding, the

outer acrosomal membrane fuse with the overlying plasma membrane, releasing the

acrosomal contents (glycohydrolases, proteases, etc.) at the site of sperm-egg

binding (acrosomal exocytosis). The acrosome is also different from the cellular

lysosome in that it contains antigens such as acrosin (Saling, 1989), acrogranin

(Anakwe & Gerton, 1990), and sperm protein AM67 (Foster et aI., 1997). Because of


36

these differences and its exocytotic properties, the sperm acrosome is considered

analogous to a secretory granule (Eddy & O'Brien, 1994).

Several biochemical and ultrastructural studies have provided evidence for the

involvement of cytoskeletal domains such as actin (Talbot & Kleve, 1978), calmodulin

(Camatini & Casale, 1987), and a-spectrin-like antigens (Virtanen et aI., 1984) in the

organisation of the acrosome. In addition, the organelle contains filamentous

structures primarily associated with the outer acrosomal membrane (Olson et al.,

1987). However the functional significance of the filamentous structures, if any, is not

yet known (Abou-Haila & Tulsiani, 2000).

Today, most researchers agree that the powerful hydrolytic enzymes

(glycohydrolases, proteases, etc) released at the site of sperm-egg binding, along

with the enhanced thrust generated by the hyperactivated beat pattern of the bound

spermatozoa (Katz & Drobnis, 1990), are the important factors regulating the

penetration of ZP and fusion of the gametes.

2.3.1 The acrosome reaction (AR)

In all mammals, sperm cells are required to fertilize oocytes, thereby providing a

haploid set of chromosomes with a paternal pattern of genomic imprinting needed for

normal development and triggering oocyte activation (Loeb, 1915; Austin, 1954;

Yanagimachi, 1994). It has long been known that successful fertilization is dependent

on the extracellular ionic environment, in large part because this can modify the

intracellular composition of gametes. The first observation was made in the sea


37

urchin when it was noted that fertilization did not occur in the absence of extracellular

Ca2+ (Loeb, 1952) due to failure of the AR to occur. After the development of

successful culture systems for mammalian gametes, it was possible to demonstrate

that mammalian sperm fertilizing ability, like that of invertebrate sperm, can be

modulated by alterations in extracellular components (Fraser, 1995).

The AR is an exocytotic process physiologically induced by ligand (ZP3)-receptor

interaction, consisting in multiple fusions between the outer acrosomal membrane

and the overlaying plasma membrane leading to the release of acrosomal enzymes

and exposure of the molecules present on the inner acrosomal membrane surface

that mediate fusion with the oolemma. The sperm acrosome is a Golgi-derived

structure forming a cap over the anterior region of the nucleus that contains many

hydrolytic enzymes and consists of an anterior cap and a posterior region called the

equatorial segment (Yanagimachi, 1994). As mentioned previously, only capacitated

sperm are physiologically able to undergo the AR in response to physiological stimuli.

It is thus conceivable that the two process, capacitation and AR, are sequentially and

functionally linked such that several of the effectors involved in mediating intracellular

signalling activated by AR start to be tuned during capacitation. For instance, the

increase of intracellular calcium levels and tyrosine phosphorylation of proteins

accompanying capacitation is also essential for the subsequent exocytosis in

response to P (Baldi et a/., 1991; Aitken et a/., 1998). The AR consists of the

development of multiple fenestrations between the outer acrosomal membrane and

the plasma membrane of the spermatozoa (Yanagimachi, 1994). This lead to the

release of the enzymatic content of the acrosome and to the exposure of the

enzymes bound to the inner membrane adjacent to the nuclear envelope


38

(Yanagimachi, 1994). In the absence of any specific stimuli only a low percentage of

human spermatozoa can undergo the AR (Leyton & Saling, 1989). It has been

suggested that self-aggregation of the sperm receptor for ZP may account for this

spontaneous acrosome reaction (Saling, 1989). A wide variety of molecules present

on the surface of the sperm have been proposed as putative candidates for the ZP3

receptor (Wassarman, 1999). The involvement of different receptors in different

species or different binding affinities, or even multiple receptors that may co-operate

in sequence to induce AR may justify this rather long list of possible candidates.

However, none of these molecules has been definitively recognised as "the sperm-

egg receptor" (Wassarman, 1999). Progesterone and a highly conserved ZP

glycoprotein termed ZP3 have been identified as natural oocyte-associated AR-

inducing ligands, and their sequential action has been shown to support the

occurrence of the physiological AR (Melendrez et ai., 1994; Roldan et ai., 1994).

There are also apparent divergences between the two pathways because the one

used by the ZP ligand involves a pertussis toxin sensitive G-protein (Franken et ai.,

1993) whereas that used by P does not (Tesarik et ai., 1993). Moreover, P also

stimulates transmembrane chloride fluxes employing a plasma membrane channel

sharing some, but not all properties with neuronal y-aminobutyric acid A (GABAA)

receptor (Wistrom & Meizei, 1993; Blackmore et ai., 1994; Shi & Roldan, 1995).

It is well known that P opens a sperm plasma membrane calcium channel and

activates phosphotyrosine kinase independently of each other (Mendoza et al.,

1995). Progesterone therefore reacts with a multiple-receptor system on the surface

and this system co-operates with that used by ZP3 to control the physiological AR.


39

Each of the respective receptors alone can eventually induce some of the AR events

and in some cases complete acrosomal exocytosis.

The AR can also be physiologically induced by P. It is present at high levels in the

cumulus matrix, surrounding the oocyte, that must be crossed by sperm in order to

reach the ZP. This steroid has been described to affect several other sperm

functions, including capacitation, motility and the priming effect on the ZP3-induced

AR through stimulation of a rapid nongenomic signalling pathway mediated by P

receptors present on the sperm surface (for further details on effects of P see Baldi et

a/., 1998; Bray et a/., 1999). P-induced AR is inhibited by 17b-estradiol through

interaction with a specific nongenomic estrogen receptor on the sperm plasma

membrane (Luconi et a/., 1999). This suggest that estradiol, which is present at

micromolar levels in the follicular fluid, may act as a physiological modulator of P

action on sperm assuring the appropriate timing of activation in the fertilization

process. Other agonists proposed to act as in vivo inducers of the AR are listed in

Table 1.


40

Table 1. Molecules that can induce the acrosome reaction in vitro.

AGONIST REFERENCEZona pellucida protein 3 (ZP3) Yanagimachi, 1994; Saling, 1989

Prostaglandin E1 Schaefer et al., 1998

ATP Foresta et al., 1993

Dibutyryl cAMP (cAMP analogues) De Jonge et al., 1991b

Progesterone & 17bOH-progesterone Baldi et al., 1998; Bray et al., 1999

Platelet-activating factor Sengoku et al., 1992; Krausz et al., 1994;

Atrial natriuretic peptide Breitbart & Naor, 1999

Epidermal growth factor Furuya et al., 1993

Serum albumin Yanagimachi, 1994

Manosylated bovine serum albumin Benoff et al., 1997a

A23187 Fênichel et al., 1989

GABA Shi et al., 1997

Forskolin De Jonge et al., 1991b

Pentoxifylline Gearon et al., 1994

1-oleoyl-2-acetylglycerol (DAG analogue) Rotem et al., 1992

Phorbol diesters (PMA, TPA) De Jonge et al., 1991 b; Lee et al., 1987

Thapsigargin Meizei & Turner, 1993


41

Many molecular mechanisms have been demonstrated to be active during AR

(Figure 5), but for many of them a precise cause-effect relationship has not yet been

defined, such that we still do not know whether they are essential for the process of

fertilization or whether they are simply associated with the process. The fundamental

difference between P- and ZP3-induced AR stands on the nature of their receptors.

In fact, in the case of ZP3, its action is mediated by receptor recruitment of G-

proteins which are not involved in a P stimulated cascade (Wassarman, 1999). One

of the first events that occur in spermatozoa following stimulation with ZP3 and P is

receptor aggregation (Saling, 1989; Tesarik & Mendoza, 1993). This is followed by a

cascade of downstream membrane and cytosolic signalling factors involved in

induction of AR (schematised Figure 5). Among them, the roles of calcium,

phospholipases and protein kinases are discussed below.


42

Na'~']ljInside

1]--=, \, pH, ••.........

ACROSOME REACTION

Figure 5. Diagram illustrating the main signal transduction pathways activated during

the process of AR in response to zona protein 3 (ZP3). Following interaction with the

agonist, aggregation of receptors for ZP3 (ZRK) induces TK activation (which

increases protein tyrosine phosphorylation) and autophosphorylation of the receptor.

Activation of phosphatidylinositol-3kinase (PI3K) has been also reported. A guanine

nucleotide binding protein (G-protein) transduces the signal interacting with

membrane-bound enzymes like phospholipase C (PLC) and AC. Activation of these

two enzymes lead to increased generation of the second messengers cyclic

adenosine monophosphate (cAMP), IP3 and diacylglycerol (DAG). A consequence of

the increase of second messengers is the activation of protein kinases such as

cAMP-dependent kinase (PKA) and Ca2+ and phospholipid-dependent kinase (PKC)

with increased protein phosphorylation. cAMP-dependent influx of sodium (Ns') has

~......... 1


43

been reported. IP3 may increase intracellular Ca2+ by liberation of the ion from

intracellular Ca2+ stores. The increase of intracellular Ca2+ consequent to activation of

ZP3 receptors is completely due to influx from the extracellular medium, is dependent

on activation of G-proteins, involves voltage-dependent Ca2+ channels and is

accompanied by an efflux of H+ which determines a rise of intracellular pH (pHi).

Partial dependence of Ca2+ -intiux from TK activation has been reported. Ca2+

dependent activation of phospholipase A2 (PLA2) and phospholipase 0 (PLO) [with

increased generation of other second messengers as arachidonic acid (AA), Iyso-

phosphatidylcholine (LC) and phosphatidic acid (PA) from membrane phospholipids

(PL)] have also been described to occur during AR. (From: Baldi et aI., 2000)

2.3.1.1 Increase in intracellular calcium during acrosome reaction

Calcium plays a central role in receptor-mediated response and membrane fusion

processes in spermatozoa (Yanagimachi, 1994). Calcium ionophores are the most

widely used non-physiological inducers of AR (Yanagimachi, 1994). Although the AR

can be induced in the absence of extracellular calcium with some agonists (Foresta

et aI., 1993; Krausz et aI., 1994; Bielfield et aI., 1994) stimulation of calcium fluxes is

one of the earlier responses activated by most stimuli that induce the AR. The shape

of the calcium wave is different during P- and ZP- stimulation. P elicits a biphasic

calcium wave, consisting of a rapid initial peak followed by a long lasting plateau

phase (Baldi et al., 1998). While ZP3 induce a slow and sustained increase of

calcium (Florman, 1994), resembling the second phase of P. However, it has been

shown, using a different methodological approach, that ZP3 may induce a rapid

increase of intracellular calcium concentrations occurring within milliseconds after

stimulation (Arnouit et al., 1999). Parallel inhibition of both AR and the second phase


44

of calcium response to P by both TK blockers and a previous administration of

estradiol, suggests the plateau phase to be responsible for agonist- induced AR

(Bonaccorsi et a/., 1995; Tesarik et a/., 1996; Bray et a/., 1999). Recently, a direct

relationship between the sustained calcium phase and AR in response to ZP3 has

been demonstrated (O'Toole et a/., 2000). Concerning P, such a relationship is less

apparent, since the percentage of sperm undergoing AR in response to the steroid is

smaller than that where a sustained response is observed (Kirkman-Brown et a/.,

2000; Kobori et a/., 2000). Although extracellular calcium depletion totally prevents

both ZP3 and P-induced intracellular calcium increase, recent data suggest the

possible involvement of intracellular calcium stores in sperm the AR (Walensky &

Snyder, 1995; Dragileva et a/., 1999; O'Toole et a/., 2000). In particular, it has been

shown that calcium entry during the sustained phase in response to ZP3 is due to

activation of store-operated channels (O'Toole et a/., 2000). While membrane

voltage-operated calcium channels of T-type have been demonstrated in mediating

ZP-induced calcium influx (Arnouit et a/., 1996; Darszon et a/., 1999), the nature of P-

stimulated calcium channels is still a matter of discussion (Garcia & Meizei, 1999;

Blackmore & Eisoldt, 1999; Patrat et a/., 2000). Calcium plays a key role in the fusion

events in the sperm membrane (Watson et a/., 1995). Using a pyroantimonate-

osmium fixation technique, the temporal and spatial location of intracellular calcium

granules was monitored during AR in ram spermatozoa (Watson et a/., 1995). Ca2+ is

initially associated with the outer acrosomal membrane. As the process progresses,

Ca2+ associates with the fusion sites between the outer acrosomal membrane and

the plasma membrane anteriorly to the equatorial segment. At later stages, Ca2+ is

localised in both post acrosomal dense lamina and on the outer acrosomal


45

membrane under the equatorial segment. These findings suggest that Ca2+ may be

implicated in the fusion process (Watson et al., 1995).

Increase of intracellular Ca2+ in response to ZP3 and P is associated with an efflux of

H+ and a rise in intracellular pH (Florman et al., 1989; Garcia & Meizei, 1996; Brook

et al., 1996). P has been shown to rapidly stimulate sodium influx (patrat et al., 2000)

and the presence of sodium in the extracellular medium is absolutely required for

induction of the AR by the steroid (Patrat et al., 2000; Garcia & Meizei, 1996). Sperm

intracellular pH may be regulated by a Na+IH+ exchanger (Garcia & Meizei, 1999) as

well as by Cr/HC03- (Holappa et al., 1999), whose presence have been

demonstrated in germ cells.

2.3.1.2 Phospholipase activation during acrosome reaction

The presence of PlA2, PLC and other phospholipases have been demonstrated in

mammalian spermatozoa (Roldan, 1998). The roles and activities of these enzymes

in human sperm capacitation and AR have been recently reviewed (Roldan, 1998). It

was also shown previously that during the AR in boar spermatozoa, the amount of

DAG and free fatty acids increases (Nikolopoulou et al., 1986). This finding was

consistent with activation of phospholipases (Roldan, 1998). In fact, increase of

[Ca2+]i stimulated by ionophores and P leads to activation of PLC in human

spermatozoa (Roldan, 1998). Such activation leads to an intracellular increase in the

amount of IP3 and DAG. Similarly, ZP3 activates the phosphoinositide specific

enzyme phospholipase Cg1 by virtue of tyrosine phosphorylation and leads to its

translocation from cytosol to particulate fractions (Tomes et al., 1996). Presence and

activation of phosphatidylcholine-specific PLC during the AR has also been


46

demonstrated (Roldan, 1998). DAG and IP3, produced by calcium-dependent

activation of PLC, may be respectively involved in the regulation of PKC activity and

release of calcium from putative sperm intracellular stores (Walensky & Snyder,

1995; Breitbart & Noar, 1999).

AR induced by ionophores and P also leads to the activation of PLA2 (Roldan, 1998).

This activation is associated with generation of lipid metabolites, such as arachidonic

acid and Iysophospholipids. Phosphatidylcholine, Iysophospholipids, and unsaturated

fatty acids, such as arachidonic acid, are potent inducers of AR (Cross, 1994;

Fleming & Yanagimachi, 1981; Kyono et al., 1984) and may be implicated in the

fusion process that occurs during AR. Moreover, LC generated from PLA2 activation

may act as a substrate for generation of platelet-activating factor (PAF) (Baldi et al.,

1993; Kumar et al., 1988). This phospholipid, which is synthesised in response to P

(Baldi et al., 1993), may further enhance AR (Krausz et al., 1994; Fukuda et al.,

1994; Huo & Yang, 2000). In vitro treatment of human sperm with PAF (1-0-alkyl-2-

acetyl-sn-glycero-3-phosphocholine) enhances penetration of zona free hamster

oocytes (Minhas, 1993). In animal studies, in vitro treatment of sperm with PAF

significantly improves fertilization rate using both intra cytoplasmic sperm injection

(ICSI) and in vitro fertilization (IVF) techniques without showing detrimental effects on

subsequent embryo development (Minhas et al., 1996; Lee et al., 1997).

2.3.1.3 Involvement of protein kinases in acrosome reaction process

During AR, activation of protein kinases (serine-threonine kinases as PKC, PKA and

protein kinase G [PKG], and TKs) is downstream to the production and/or activation

of early second messengers. Early evidence for the involvement of PKA in the AR,


47

demonstrated that AC activity and cAMP generation increase during this process (De

Jonge et ai., 1991a). Adenyl cyclase stimulators, such as forskolin, and the cAMP

analogue dibutyryl cAMP, induce AR in a dose-dependent manner in mammalian

sperm (Anderson et ai., 1992; Garde & Roldan, 2000; Harrison & Meizei, 2000;

Kobori et ai., 2000). In addition, PKA inhibitors have been reported to suppress the

AR induced by P (Harrison & Meizei, 2000), although controversial results are

present in the literature concerning an increase of cAMP synthesis in response to P

(Parinaud & Milhet, 1996; Schaefer et al., 1998). Taken together, these findings

suggest that PKA activation may be involved in the AR process (Baldi et al., 2000).

The involvement of PKC in the process of AR is still under debate. Although

activators such as phorbol esters and synthetic DAG induce the AR, PKC activity in

human sperm is very low and the identity of PKC substrates is still under

investigation. Induction of AR by solubilized ZP was partially reduced by pre-

treatment with inhibitors of PKA, PKC and PKG tested separately, while combinations

of them caused a significantly greater inhibition (Bielfield et ai., 1994). These results

suggest a concomitant role for PKA, PKC and PKG in the human ZP-induced AR

(Bielfield et ai., 1994). Both P (O'Toole et ai., 1996; Bonaccorsi et ai., 1998) and ZP

(Liu & Baker, 1997) increase sperm PKC activity. Such activation is downstream to

calcium influx (P (Bonaccorsi et ai., 1998), since it can be prevented by calcium

channel blockers (O'Toole et ai., 1996).

ZP3 induced an increase in tyrosine phosphorylation of sperm proteins (Ley ton &

Saling, 1989; Burks et al., 1995). A major tyrosine phosphorylated protein in

mammalian spermatozoa was 95-97 kDa (Leyton & Saling, 1989; Burks et ai., 1995).


48

Proteins in this molecular weight range appear to undergo phosphorylation during

capacitation (see above) and in response to P (Baldi et a/., 1998). Recombinant

human ZP3 produced by genetic engineering has been demonstrated to induce AR

and tyrosine phosphorylation of the 95-110 kDa protein in human sperm (Brewis et

a/., 1998), perfectly mimicking the physiological action of this glycoprotein. Known

inhibitors of TK, such as genistein and tyrphostin-47, block ZP3-induced AR (Leyton

et a/., 1992). Both the TK inhibitors tyrphostin A48 and pertussis toxin suppress ZP3-

induced calcium influx in mouse spermatozoa (Bailey & Storey, 1994). Using a

similar pharmacological approach, Tesarik et al. (1993) and Luconi et al. (1995)

showed involvement of TKs in the P-mediated AR. Activation of TK also seem to be

involved in the plateau phase of increase in the amount of intracellular Ca2+ in

response to P (Bonaccorsi et a/., 1995; Tesarik et a/., 1996). The mechanisms

through which TKs are activated in sperm during the process of AR are still under

investigation. Although during capacitation activation of the AC/cAMP/PKA pathway

appears to be highly involved, activation of this pathway in response to stimuli that

induce AR is less clear (Parinaud & Milhet, 1996; Schaefer et a/., 1998). The

involvement of reactive oxygen species, generated by sperm in response to stimuli

that induce AR, in tyrosine phosphorylation has been suggested by a few

researchers (de Lamirande et a/., 1998; Fisher et a/., 1998).

Recent data (Fisher et a/., 1998) indicate the involvement of PI3K in the AR

stimulated by agents that mimic ZP3. These include mannose-BSA and polyclonal

antibodies raised against the p95 protein (indicated as the possible sperm ZP3

receptor; see ZRK above), but not by P or ionophores (Baldi et a/., 2000).


49

2.4 Motility

Human spermatozoa emerge highly differentiated from the testes, but as for all other

eutheria, they are motionless or feebly motile. This immotility is apparently due to the

"immaturity" of the plasmalemma, as demembrated spermatozoa can be induced,

under appropriate conditions to move almost as actively as mature spermatozoa from

the cauda (Mohri & Yanagimachi, 1980). The spermatozoa continue to develop

during their passage through the epididymis. Only at ejaculation, when they are

mixed with the secretions of the accessory glands, do spermatozoa undergo motility

activation, acquiring a mature motility pattern and fertilizing ability.

Sperm motility plays an important role in transport of the spermatozoa in the female

reproductive tract before fertilization (Yanagimachi, 1969). Sperm motility is a

complex phenomenon, the understanding of which requires integration of cell biology

with reproductive physiology, biochemistry, biophysics and clinical andrology. The

spermatozoon is an intricate motile cell, whose motility depends on a flagellum that

develops the propulsive force for swimming. The likelihood of achieving a pregnancy

increases with decreasing proportions of immotile spermatozoa and with increasing

quality of sperm progression (Bostofte et al., 1983; Bostofte et al., 1984).

Over the years a plethora of terminology has appeared to describe the various

movement characteristics of motile spermatozoa. Consensus on these parameters

was reached at the "Automated Sperm Motility Analysis" workshop held at the

American Society of Andrology's Annual Meeting in Houston (Tx, USA) during March

1988 (Mortimer 1990). These parameters have now been standardised and are

currently used in motion analysis (Owen & Katz, 1993). These parameters include:


50

Curvilinear velocity (VCL): The VCl (urn/s) is the time-average velocity of a sperm

head along its actual curvilinear path.

Straight line velocity (VSL): The VSl (um/s) is the time-average linear velocity of a

sperm head along the straight line between the start and the end of the observed

track.

Average path velocity (VAP): The VAP (!-ImIs) is the time-average velocity of a sperm

head along its average path. This path is computed by smoothing the actual path

according to algorithms in the CASA instrument.

Amplitude of lateral head displacement (ALH): The AlH (urn) is the magnitude of

lateral displacement/deviation of a sperm head about its average path. It can be

expressed as a maximum or an average of such displacements.

Beat-cross frequency (BCF): The BCF (Hz) is the average rate at which the sperm's

curvilinear path crosses its average path.

Straightness (STR): The STR (%) is the ratio of VSLlVAP and measures the

straightness of the average path.

Linearity (Lin): The Lin (%) is reported as the ratio of VSLlVCl and measures the

linearity of the curvilinear path. The value range from 0 to 100 with a value of 100

representing cells swimming in a straight line.

All these motility parameters, as depicted in Figure 6, give a clear indication of

movement characteristics of spermatozoa (WHO, 1999).


51

vel

Figure 6: Motion parameters of a single sperm track, where ALH = amplitude of

lateral head displacement; BGF = beat-cross frequency; VAP = average path

velocity; VGL = curvilinear velocity and VSL = straight line velocity.


52

2.4.1 Factors influencing sperm motility

Enhanced sperm motility values have been associated with improved conception

rates (Edvinsson et a/., 1983). In this regard, efforts have been directed towards

improvement of poor motility of sperm as well as the inducement of motility of

immotile cells. The practice of mixing sperm with a motility stimulating agent for

improving the successful rate of artificial insemination has been reported (Yovich et

al., 1988).

2.4.1.1 Cyclic adenosine mono-phosphate (cAMP)

Cyclic nucleotides play a major role in sperm capacitation, metabolism, AR and

motility (Tash & Means, 1983). This was first observed when it was noted that

caffeine and other methyl-xanthines, which act as cyclic nucleotide PDE inhibitors

and associated with an increase in intracellular cAMP, can stimulate sperm motility

and metabolic activity (Hoskins, 1973).

Garbers et al. (1971) first showed that the addition of dibutryl cAMP stimulated the

motility of bovine caudal epididymal spermatozoa. cAMP was shown to have effects

on the metabolism and motility of sperm (Tash & Means, 1982). Since then the cAMP

system of spermatozoa has been extensively studied.

Elevation of intracellular cAMP levels appear to be related to:

... the acquisition of the potential for motility during epididymal maturation;

... the actual initiation of motility upon ejaculation; and

...the surge of increased motility associated with capacitation.


53

The cAMP content of diluted spermatozoa increases two fold as sperm traverse the

epididymis (Hoskins et ai., 1974). This seems reasonable since the intracellular

concentrations of cAMP correlates directly with the degree of motility (Hoskins &

Casillas, 1975). Furthermore, a 25% decrease in cAMP levels is observed as washed

bovine caudal sperm become immotile as compared to the initial motility (Hoskins et

ai., 1974). During ejaculation, as bovine caudal epididymal spermatozoa are mixed

with secretions from the accessory glands, cAMP levels double within 30 seconds

(Cascieri et ai., 1976). A similar rise in cAMP concentration has been reported in

hamster spermatozoa (Morton et ai., 1974).

cAMP has also been implicated with sperm motility during capacitation and AR

(Rosado et ai., 1974). Vigorous motility is commonly observed concomitant with

capacitation and is believed to be essential for penetration of the egg following the

AR (Yanagimachi, 1970). Reyes et al. (1977) showed that the addition of exogenous

cAMP to rabbit or human spermatozoa more than doubled the rate of capacitation in

vitro.

cAMP appears to activate cAMP dependant kinase, which phosphorylates key

proteins that are required for motility (Lindemann & Kanous, 1989). Deactivation

occurs when these proteins are dephosphorylated by phosphoprotein phosphatase.

The phosphorylation of several integral axonemal proteins has been reported to be

cAMP-dependant (Tash et al., 1984; Horowitz et al. 1988).


54

2.4.1.2 Adenylate cyclase

Mammalian sperm possess both AC and POEs, which are involved in the regulation

of the intracellular concentration of cAMP (Bhatnagar et al., 1982) and also possess

cAMP-dependant protein kinase through which the physiological action of cAMP is

mainly mediated. Pariset et al. (1983) reported that there is a positive correlation

between the motility index and the cAMP-dependant protein kinase activity of human

spermatozoa.

A significant positive correlation was found between sperm motility and cAMP content

and AC activity in human spermatozoa (Ishikawa et al., 1989). These results suggest

that sperm motility is controlled by the AC activity in spermatozoa and that the

disturbance of sperm motility in infertile men is probably caused by reduced activity of

AC.

2.5 Summary

Mammalian sperm are not immediately fertile upon release from the male

reproductive tract, despite their ability to exhibit vigorous motility. They require a

species dependent period of time during which they undergo a series of changes,

collectively referred to as capacitation (Austin, 1952; Yanagimachi, 1994), that are

needed for cells to become fully competent to fertilize an oocyte. When capacitated,

mammalian sperm can (i) express hyperactivated motility, the very vigorous, thrusting

pattern of motility that is needed for penetration of the oocyte investments and (ii)

interact with the oocytes (including cumulus cells, folucular fluid and ZP) to undergo

the AR. Capacitation is a unique feature of mammalian sperm that occurs in the

female tract and is essential for fertilization. During capacitation, sperm acquire


55

hyperactive motility and the ability to undergo a regulated AR. Capacitation is

accompanied by changes in lipid composition of the plasma membrane as well as by

protein post-translational modifications regulated by several signalling pathways.

Studying in vitro capacitation has allowed characterisation of a number of

biochemical events that occur in human spermatozoa. It is not clear whether the

same or similar events occur during capacitation in vivo. P, PAF and other effectors

present in the follicular fluid and/or cumulus matrix, could facilitate capacitation or

prime the AR in vivo. For instance, the clinical significance of P effects has been

shown by several studies showing a significant correlation of response to P with

sperm fertilizing ability as well as reduced or absent response to P in infertile subjects

(Baldi et a/., 1998; Bray et aI., 1999). Moreover, treatment of human sperm with P

has been shown to enhance hamster oocyte penetration (Aitken et aI., 1996). These

studies indicate that sperm P receptor, as well as the one that mediates ZP3

stimulation, is involved in the process of fertilization representing a possible target for

developing pharmacological strategies to potentiate sperm fertilizing ability in

assisted reproductive techniques or contraceptive molecules. Functional Preceptors

on the human sperm surface have been recently characterised (Luconi et al., 1998;

Falkenstein et aI., 1999). Sequencing of the receptor and cloning of the encoding

gene will represent next steps in such characterisation.

The AR is an exocytotic event that promotes interaction and penetration through the

ZP and confers fusogenic properties on the remaining plasma membrane in the

sperm head (Yanagimachi, 1994).


56

It has been suggested that the importance of capacitation may actually be to prevent

sperm from becoming fertile too quickly, given that spermatozoa are deposited into

the lower regions of the female reproductive tract and still need to travel some

considerable distance in order to reach the site of fertilization (Bedford, 1983). The

AR must therefore also be accurately timed to ensure fertilization, since a premature

AR leads to the loss of ZP recognition sites from the sperm surface and thus impairs

sperm-zona pellucida binding (Franken et aI., 1993). On the other hand inability of

zona bound spermatozoa to activate the AR also prevents zona penetration.

Although work emanating from multiple laboratories is leading to a better

understanding of capacitation, AR and motility, several of the proteins involved in

these processes remain to be characterised.


57

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"All life is an experiment."

- Ralph Waldo Emerson -


87

CHAPTER 3

MATERIALS AND METHODS

3.1 Introduction

The material and methods used for each study is outlined in the individual articles as

presented in the following chapters. The methods differed slightly between the four

studies, however the basic, common and shared materials and procedures used will

subsequently be discussed.

3.2.1 Preparation of Human tubal fluid (HTF) culture medium

This simple culture medium was developed from the composition of human oviduct

fluid (Quin et al., 1985) and has proved useful for in vitro studies and clinical

applications including IVF, GIFT and lUI. Chemicals used should be at least of

analytical grade and the water to be used of tissue culture grade. The recipe for the

preparation of HTF are as follows:

1. Dissolve the following chemicals in about 600ml tissue culture grade water in a

1000-ml volumetric flask: 5.938g NaCI; 0.350g KCI; 0.049g MgS04.7H20; 0.050g

KH2P04; 2.100g NaHC03; 0.036g Na pyruvate; 0.501g Glucose; 0.003g Phenol

red; 3.136 ml Na lactate (60% syrup)

2. Separately dissolve 0.300g CaC12.2H20 in 100mi tissue culture grade water and

add slowly to the rest.

3. Add Penicillin/Streptopen (75mg)

4. Make up to 1OOOmlwith additional culture grade water and mix thoroughly.

5. Adjust pH to 7.5-7.6

6. Check that osmolarity is between 280-290 mOsm.


88

7. Filter-sterilise into plastic containers under positive pressure.

8. Store at 4°C

9. Warm to 37°C before use

Add 3% BSA if the medium is to be used as a capacitation medium.

3.2.2 Semen collection

Semen samples were obtained from patients attending the in vitro fertilization

programme at Tygerberg hospital and from healthy donors. All semen samples were

collected by masturbation after 2-3 days of sexual abstinence (WHO, 1999).

Collections were made in sterile plastic containers after which the semen was

allowed to liquify for 30 minutes at 37°C.

3.2.3 Oocyte collection and storage

All oocytes used were nonliving, with no developmental potential. Oocytes were

obtained from ovarian tissue that was collected post mortem. This procedure fully

complies with the legal and ethical guidelines of the South African Medical Research

Council. Ovarian tissue was excised within 24 hours of death and subsequently

macerated/manually dissected (Overstreet et al., 1980) and flushed with phosphate

buffered saline (PBS, GIBCO, Grand Island, NY). The PBS flushing was stored at

4°C for no longer than 48 hours. Zona intact oocytes denuded of granulosa cells

were recovered under microscope and placed directly into a 1.5M solution of MgCI2

and stored at 4°C under mineral oil (E.R. Squib & Sons Inc, Princeton, NJ) for

immediate use. Oocytes were also stored in a dimethylsulfoxide/sucrose (DMSO)

solution at -196°C in liquid nitrogen (Hammit et ai., 1991). Twenty four hours prior to


89

each test, oocytes were removed from storage and thawed at 37°C. Retrieved

oocytes were placed in 0.25M sucrose and 3% BSA in HTF.

3.2.4 Solubilized zona pellucida preparation

On the day of the experiment, 20 oocytes were placed in a drop on a petri dish with a

glass drawn Pasteur micro pipette, after which the HTF was removed under

microscopic vision (Olympus SZ40; Wirsam Scientific, Cape Town, South Africa),

leaving only the 20 oocytes on the petri dish. A total volume of 5JlIof 10mM HCl was

then added to the oocytes on the petri dish in order to dissolve the zonae at room

temperature. Solubilization of the ZP was microscopically observed and controlled

(45-60min). Following solubilization, the reaction was neutralised by adding 5JlI of

10mM NaOH rendering a final volume of 10Jll. Solubilized ZP solutions with

concentrations of 2 ZP/JlIwere stored up to one week at 4°C.

3.2.5 Assessment of the acrosome reaction

Spermatozoa from the different experiments were fixed on separate spots of spotted

slides (MAGV, Germany, XER 201B) and air-dried. Each spot was flooded with

fluorescein-labeled Pisum Sativum agglutinin solution (FITC-PSA; 125Jlg/ml; l-0770;

Sigma, Cape Town, South Africa) and left in the dark for 30 minutes. After 30 minutes

of incubation the excess FITC-PSA was rinsed of in a beaker containing distilled

water. Mounting medium and a cover slip was placed on the slides and then

evaluated within 24 hours under a fluorescence microscope. Two individuals using

the blind scoring method microscopically scored a minimum of 200 spermatozoa for

each different point. The following staining patterns were evaluated as acrosome

reacted spermatozoa; (i) patchy staining on acrosomal region, (ii) distinct staining in


90

the equatorial region occurring as an equatorial bar and (iii) no staining over the

entire sperm surface. Spermatozoa with patchy FITC-PSA staining were classified as

a population of sperm where the acrosome reaction was initiated and all were

classified as acrosome reacted (Figure 1).

Unreacted Patchy Equatorial Reacted

All classified as "Reacted"

Figure 1:Patterns recorded during FITC-PSA acrosome staining procedures.


91

3.2.6 Hemizona binding assay

This functional assay assesses tight binding of sperm to the outer surface of the zona

pellucida hemisphere. It allows a controlled comparison of sperm-zona binding

between control and test spermatozoa, as the zonae surfaces are functionally equal.

3.2.6.1 Bisecting of oocytes

A complete micromanipulation system (Narishige, Tokyo, Japan) was used for

bisecting the oocytes. An inverted phase-contrast microscope (Nikon Diaphot,

Garden City, NY) was equipped with a pair of Narishige micromanipulators (model

MO 102), the connecting tubing filled with mineral oil, and a #11 microscalpel blade

attached to the manipulator. A 100mm petri dish (Falcon #25382, Falcon plastics,

Oxnard, CA) served as the cutting chamber. Culture medium was poured into the

dish to a depth of 3mm. Horizontal grooves were cut into the plastic in order to

provide support in holding the egg while cutting. The egg was transferred to the

working area of the dish using a finely drawn Pasteur glass pipette. Using a total

magnification of 200x, the blade was centred and then slowly lowered, first partially

flattening the egg and then finally initiating a midline cut into the zona. A further

lowering of the blade, along with 1 to 2 side to side excursions, produce two cleanly

cut hemizonae. Vigorous pipetting was used to then dislodge the dense ooplasma

inside each hemizona. Only one egg was cut at a time to ensure that matched

hemizona remained paired for subsequent sperm binding tests. Each hemizona pair

was placed in a 501J1droplet of medium in a petri dish, covered with mineral oil and

stored at 4°C.


92

3.2.6.2 Competitive sperm-binding to the hemizona

A 501J1sperm droplet containing O.5x106 spermimi from both the test and control

samples were placed in a marked tissue culture dish. One hemizona was placed in

the one droplet and the matching hemizona in the other (Figure 2). The droplets were

covered with mineral oil in order to prevent evaporation and dehydration of the sperm

droplets and co-incubated at 37°C and 5% C02 for 4 hours. After co-incubation, the

hemizona from each droplet was transferred into culture medium (HTF) droplets

respectively. The hemizonae were then rinsed by vigorously pipetting five times with

a finely drawn glass pipette in order to dislodge loosely associated spermatozoa. The

number of spermatozoa firmly bound to the outer surface of each hemizona was

counted under phase contrast microscopy (Nikon TMS-F, Research Inst.

Johannesburg, South Africa) at a 200x magnification. The number of sperm bound in

the test sample was divided by the number of sperm bound in the control sample and

expressed as a percentage to give a hemizona index (HZI).


93

HEMIZONA SPERM BINDING ASSAY

Human oocyte.. ..~ rri\ Oocyte is bisected into~ ~ 2 identical Hemizonae

Jl' "

Test Control

Hemizonae are incubated in test and~ spermpopulationsfor4&

The number of spermfirmly bound to Hemizona are counted and sperm-zona binding

is expressed as Hemizona index

Zona bound sperm (test) x 100Binding index:Zona bound sperm (control)

Figure 2: The competitive hemizona sperm binding assay.


94

3.2.7 Computer assisted semen analysis (CASA)

Sperm motility/kinematics were determined with the Hamilton-Thorne IVOS analyser

(Hamilton-Thorne Research, Beverly, MA) with standard set-up parameters. The

analyser settings were as follows: 30 frames/60 Hz; minimum contrast, 80; minimum

cell size, 2; minimum static contrast, 30; low average path velocity (VAP) cut-off, 5

mts; low VSL cut-off, 11 mts; head size, nonmotile, 3; head intensity, nonmotile, 160;

static head size, 1.01-2.91; static head intensity, 0.60-1.40; slow cells not motile;

magnification, 2.01, and temperature, 3rC.

Low sperm counts «10 x 106/mL) were confirmed by manual analysis, while high

counts «50 x 106/mL) were diluted 1:1 with HTF medium for accurate determination

of count by the CASA (IVOS) system.

3.2.8 Statistical Analyses

All statistical evaluations and tests were carried out using GraphPad Prism 2.01.

Data are expressed as mean±SE. Student's t-test for paired data was used to

compare the results of all the acrosome and motility studies, while Pearson's test was

used to perform correlation tests. The Mann-Whitney U test for nonparametrie data

was used to compare binding results. A Hemizonae Index was also calculated for

matched hemizonae assay results and expressed as a mean percentage where HZI

- Test sperm bound to hemizona/ 100) P I I I th- Control sperm bound to hemizona X . -va ues equa or ess an

0.05 were considered statistically significant.


95

References

Hammit DG, Syrop CH, Walker DL, Bennet MR (1991). Conditions of oocyte storage

and use of non inseminated, non-fertilized oocytes for the hemizona assay.

Fertil SteriI60:131-136.

Overstreet JW, Yanagimachi R, Katz OF, Hayashi K, Hanson FW (1980) Penetration

of human spermatozoa into the human zona peJlucida and the zona-free

hamster egg: a study of fertile donors and infertile patients.Fertil Steril.

33(5):534-42.

Quin P, Kerin JF, Warnes GM (1985). Improved pregnancy rate in human in vitro

fertilization with the use of a medium based on the composition of human tubal

fluid. Fertil Steril 44:493-498.




92.


"There is one thing even more vital to

science than intelligent methods; and that

is, the sincere desire to find out the truth,

whatever it may be."

- Charles Sanders Pierce -


96

CHAPTER4

THE ZONA PELLUCIDA-INDUCED ACROSOME REACTION OF

HUMAN SPERMATOZOA INVOLVES EXTRACELLULAR SIGNAL-

REGULATED KINASE ACTIVATION

ANDROLOGIA 2001 Vol 33 no. 6 pp337-342

SS du Plessis1,*, C page1 and DR Franken2

1Department of Medical Physiology and Biochemistry, University of Stellenbosch,

P.O.Box 19063, Tygerberg, 7505, South Africa.

2Department of Obstetries and Gynaecology, University of Stellenbosch, Tygerberg

Hospital, Tygerberg, 7505, South Africa.

Running head: Role for ERK during acrosome reaction

Keywords: spermatozoa, acrosome, ERK, zona pellucida

'Corresponding author. SS du Plessis, Department of Medical Physiology and

Biochemistry, University of Stellenbosch, P.O.Box 19063, Tygerberg, 7505, South

Africa; Tel.: +27-(0) 21-938 9388; fax: +27-(0) 21-938 9476; e-mail:

[email protected].


mailto:[email protected].

97

Summary

Extracellular signal-regulated kinases (ERK), a member of the family of mitogen-

activated protein kinases (MAPK) are cytoplasmic and nuclear serine/threonine

kinases involved in signal transduction of several extracellular effectors. Recent

evidence indicates the presence of p21Ras and the phosphorylation of ERK-1 and

ERK-2, suggesting the occurrence of the Ras/ERK cascade in mammalian

spermatozoa. We report here on the biological role of ERKs during the acrosome

reaction, on stimulation with zona pellucida (ZP), in human spermatozoa. The

mitogen-activated protein kinase inhibitor PD098059 was used as a pharmacological

tool to study the involvement of extracellular signal-regulated kinases in the induction

of the acrosome reaction in human spermatozoa. This compound significantly

inhibited both the ZP- and A23187-induced acrosome reactions. These results

suggest that ERKs are involved in the signal transduction pathway through which ZP

stimulation works during the process of fertilization.

Introd uction

The development of the fertilization-competent state of the spermatozoon occurs

through a series of poorly understood processes including capacitation and

acrosomal reaction (AR). During the process of capacitation spermatozoa acquire the

ability to fertilize the oocyte. Capacitation is characterised by a series of profound

membrane and metabolic transformations in spermatozoa that increase their ability to

respond to physiological stimuli of the acrosome reaction (Yanagimachi, 1994) [1].

The AR is a specialised exocytotic event consisting of multiple fusion's and

fenestration's of the outer acrosomal membrane and the overlying plasma membrane

that lead to subsequent release of the acrosomal enzymes that help the spermatozoa


98

to penetrate the zona pellucida (Yanagimachi, 1994). These two activational

processes appear to be regulated by intracellular signalling systems similar to those

utilised by somatic cells (Kopf et al., 1995). Although the precise signal transduction

pathways have not been fully elucidated (Baldi et al., 1996) it is known that both

processes are characterised by increases in intracellular calcium concentrations and

phosphorylation of proteins including that in tyrosine residues (Leyton & Saling, 1989;

Carr & Acott, 1990; Naz et aI., 1991; Duncan & Fraser, 1993; Burks et aI., 1995;

Luconi et aI., 1995; Visconti et aI., 1995; Luconi et al. 1996).

Several reports from studies in somatic cells focused attention on the presence and

activation of the mitogen-activated protein kinase (MAPK) family of kinases in

response to ligands binding both G-protein coupled receptors as well as protein

tyrosine kinases (PTK) receptors. The MAPK's are a family of serine/threonine

kinases with multiple membrane, cytosolic and nuclear substrates, the activation of

which lead to an array of responses. This include the activation of (gene transcription

via translocation of MAPK into the nucleus) genes encoding for protective proteins in

response to stress, cell proliferation, cell differentiation, apoptosis and exocytosis

(Offermans et aI., 1994; Page & Doubell, 1996; Canman & Kastan, 1996). It is also

known that the activation of the MAPK family of kinases can be up- or down

regulated through crosstalk with other signalling pathways i.e. protein kinase A

(PKA), protein kinase C (PKC) and PTK in many cell types (Bogoyevitch et aI., 1994;

Burgering & Bos, 1995). Extracellular signal-regulated kinases, ERK-1 (p44 MAPK)

and ERK-2 (p42 MAPK), are the members of this family studied the most in somatic

cells. The presence and biological activity of p21 Ras has recently been

demonstrated in human spermatozoa (Naz et al., 1992). Recent characterisation of a


99

boar sperm protein kinase with characteristics similar to those of ERK-2 (Berruti,

1994), as well as demonstrating the presence of ERK's in human spermatozoa

(Luconi et ai., 1998) and the activation thereof during in vitro capacitation lead to the

hypothesis that the Ras/Raf/ERK cascade may be involved in the capacitation and/or

AR in human spermatozoa during zona pellucida (ZP) stimulation.

As of yet very little is known about ZP-mediated sperm signal transduction in the

human, due, for the most part, to an inability to obtain sufficient quantities of human

ZP for experimental purposes. The human ZP has been shown to bind human

spermatozoa and to induce the AR of spermatozoa (Cross et ai., 1988; Morales et

ai., 1989).

The present study aimed to determine the regulatory role of MAPK and in particular

ERK during the acrosome reaction in human spermatozoa.

Materials and Methods

Preparation of sperm sam pies

Semen samples were collected from normozoospermic donors by masturbation after

2-3 days of sexual abstinence. Semen samples were analysed according to the

World Health Organisation criteria (WHO, 1992) together with strict sperm

morphology assessment (Kruger et ai., 1986). Motile sperm fractions were collected

from samples using a slightly modified double-wash swim-up technique. Retrieved

sperm samples were resuspended in synthetic human tubal fluid medium (HTF)

(Quin et ai., 1985) supplemented with 3% bovine serum albumin (BSA; Seravac,


100

Cape Town, South Africa) to a sperm concentration of 10x106 cells/ml. Before the

onset of AR studies, sperm samples were allowed to capacitate at 37°C in 5% CO2

for 3 hours in HTF-BSA. Prepared sperm samples were incubated in the presence or

absence of the MEK-inhibitor, PD098059 (P-215, Sigma, Cape Town, South Africa)

at a concentration of 50/J.Mfor 90 minutes at 37°C.

Preparation of solubilized ZP

Oocytes were retrieved from post mortem derived ovarian material. (The project fully


Council.) Oocytes were stored in a dimethylsulfoxide/sucrose solution at -196°C in

liquid nitrogen (Hammit et al., 1991). Twenty four hours prior to each test, oocytes

were removed from storage and thawed at 37°C. Retrieved oocytes were placed in

0.25M sucrose and 3% BSA in HTF. On the day of the experiment, 20 oocytes were

placed in a drop on a petri dish, after which the HTF was removed under microscopic

vision (Olympus SZ40; Wirsam Scientific, Cape Town, South Africa), leaving only the

20 oocytes on the petri dish. A total volume of 5/J.1of 10mM HCl was then added to

the oocytes on the petri dish; solubilization of the ZP was microscopically observed

and controlled. Following solubilization, 5/J.1of 10mM NaOH was added to the

solubilized ZP, to render a final zona volume of 10/J.1containing 2 ZP//J.1.The final ZP

concentration, after the addition of spermatozoa, was 0.67 ZP//J.1.

Acrosome reaction studies

Acrosomal statuses of spermatozoa stimulated with (i) 10/J.MA23187 (C-7522,

Sigma, Cape Town, South Africa) for 20 minutes and (ii) 0.67ZP//J.1for 60 minutes in

either the presence or absence of PD098059 (pre-treatment) were determined


101

according to procedures published elsewhere (Cross et ai., 1988; Morales et ai.,

1989, Franken et al., 2000) and compared to that of control samples. Control

samples were allowed to spontaneously acrosome react.

Prior to aspiration into Teflon tips during the micro-assay (Franken et al., 2000), the

sperm/ZP suspensions were gently mixed in a well of a 60 well micro-titre plate

(Microtest plate cat No. P43, Laboratory and Scientific, Cape Town South Africa).

Aspirating HTF droplets into both sides of the Teflon tip sealed off the sperm

suspensions and prevented evaporation from the tip. Each sperm/ZP suspension

was separated from the HTF droplets by air bubbles on both sides.

Progressive motility for both acrosome reaction techniques was monitored before and

after the incubation periods. Sperm droplets were carefully placed on separate spots

of spotted slides (MAGV, Germany, XER 201B) and immediately evaluated for

percentage live sperm under inverted phase contrast microscope (Nikon TMS-F,

Research Inst. Johannesburg, South Africa). Sperm samples were obtained after

swim-up and only samples with a progressive motility of more than 80%, according to

the World Health Organisation criteria (WHO, 1999), were subsequently used in the

experiments.

Spermatozoa from the different experiments were fixed and air-dried, after which

acrosomal status was determined using fluorescein-labeled Pisum Sativum agglutinin

(FITC-PSA; 125f.!g/ml; L-0770; Sigma, Cape Town, South Africa). Two individuals

using the blind scoring method scored a minimum of 200 spermatozoa for each

different point. The following staining patterns were evaluated as acrosome reacted


102

spermatozoa; (i) patchy staining on acrosomal region, (ii) distinct staining in the

equatorial region occurring as an equatorial bar and (iii) no staining over the entire

sperm surface. Spermatozoa with patchy FITC-PSA staining were classified as a

population of sperm where the acrosome reaction was initiated and all were classified

as acrosome reacted.

Statistical Analysis

The percentage acrosome reacted sperm were compared using Student's paired t

test. p-values equal or less than 0.05 were considered statistically significant.

Results

Acrosome reactions induced spontaneously were completely insensitive to the pre-

treatment of spermatozoa with P0098059 and the mean percentage of acrosome-

reacted spermatozoa remained. As expected, A23187 stimulation induced the AR

and the mean percentage of acrosome-reacted spermatozoa increased with 18.8%,

which was significantly higher than that of the control values (p<0.05). However, after

PO pre-treatment, A23187 stimulation did not induce the AR reaction significantly

more than control values (Figure 1).

ZP stimulation also induced the AR and increased the mean percentage of acrosome

reacted spermatozoa significantly by 9.2% (p<0.05). P0098059 pre-treatment of

spermatozoa resulted in a complete inhibition of ZP stimulated AR and the mean

percentage of acrosome reacted spermatozoa stayed at basal values (13.5%)

(Figure 2).


103

25.0

20.0 -

15.0 -r::t::« 10.0 -::::e0

5.0*

0.0 I-5.0 PD

*

Figure 1. Influence of the MEK-inhibitor P0098059 (PO) on the acrosome reaction

(Mean±SE) mediated by A23187 (difference between acrosome reaction and the

spontaneous percentage acrosome reaction of 23.9±2.2%). (n=10)

* p<O.05 compared with A23187

Capacitation seems to have been completed before the incubation of the

spermatozoa with the MEK-inhibtor (PD098059) since these cells were able to

acrosome react in response to both ZP and A23187 stimulation (results not shown).


104

25.0

20.0 -

15.0a::<C 10.0

5.0 -

0.0 --~-

-5.0 -

* *

___PO _ ZP- :{-PD +_ZP

I

Figure 2. Influence of the MEK-inhibitor P0098059 (PO) on the acrosome reaction

(Mean±SE) mediated by ZP (difference between acrosome reaction and the

spontaneous percentage acrosome reaction of 13.5±2.0%). (n=3)

* p<O.001 compared with ZP

Discussion

The present paper shows that ERK activation plays a biological role in the ability of

human spermatozoa to undergo the acrosome reaction, since the ability of these

cells to acrosome react in response to ZP and the calcium ionophore, A23187, is

strongly inhibited in the presence of the MAPK (ERK) cascade inhibitor, PD098059.

To understand the signalling pathways through which both receptor induced (ZP3

binding to the ZP membrane receptor) and non-receptor induced (opening of calcium

channels by an ionophore) mechanisms could elicit the acrosome reaction, a

modified version of the recently hypothesized scheme of Breitbart and Spungin


105

(1997) will be referred to (Figure 3). The ZP3 glycoprotein binds to at least two

receptors in the plasma membrane. One receptor is a Gj-coupled receptor that

activates phospholipase C (PLC) ~1. The other receptor is a tyrosine kinase receptor

(TKR) coupled to PLCy Binding to the Gj-coupled receptor would regulate adenylate

cyclase (AC) leading to the elevation of cAMP and PKA activation. PKA activates a

voltage-dependent Ca2+ channel in the outer acrosomal membrane, which releases

Ca2+ from the interior of the acrosome to the cytosol. This relative small rise in [Ca2+]j

could result in the activation of PLCy The products of phosphatidyl-inositol

biphosphate (PIP2) hydrolysis by PLCf11 and PLCy, diacylglycerol (DAG) and

inositoltrisphosphate (IP3) lead to PKC translocation to the plasma membrane and its

subsequent activation. This increase in [Ca2+]j can be mimicked by the addition of a

calcium ionophore (e.g. A23187), which will also result in the activation of PLCy, and

PKC activity. PKC opens a voltage-dependent Ca2+ channel in the plasma

membrane, increasing the [Ca2+]1 even more. PKC activation also results in the

activation of ERK, through its ability to phosphorylate and activate an upstream

mediator of the ERK cascade, Raf. Raf will activate and phosphorylate the ERK

kinase, MEK, which in turn activates ERK. The Gj and TKR can also activate a

Na+/H+ exchanger, leading to alkalization (pH increase) of the cytosol. The increase

in [Ca2+]j and pH will lead to membrane fusion and acrosomal exocytosis.


106

ZP3

Plasma membrane

Ras

Outer acrosomal membrane

Figure 3. Possible interactions between the different signal transduction pathways

invoked during the acrosome reaction. (ZP3 = zona pellucida glycoprotein; R = G;-

coupled receptor; TKR = tyrosine kinase receptor; dashed lines = hypothesised

activation of ERK) (Modified from Breitbart & Spungin, 1997)

In support of this hypothesis, it was previously shown in our laboratories that the ZP-

induced AR appears to be mediated through a G-protein-mediated signal

transduction process after functional inactivation of the Gi-protein receptor by


107

pertussis toxin (Bastiaan et ai., 1999). Aitken et al. (1996) have also shown that the

addition of the tyrosine kinase inhibitor, genistein, inhibited capacitation, probably by

inhibiting the protein tyrosine kinase ZP receptor. In agreement with the hypothesis,

Luconi et al. (1998) also showed that in vitro capacitation stimulates a sustained and

concomitant increase in tyrosine phosphorylation and kinase activity of ERKs,

indicating the activation of these enzymes during capacitation. It was also shown that

the ability of human spermatozoa to undergo the AR in response to progesterone

were strongly inhibited when capacitation was performed in the presence of the

MAPK cascade inhibitor PD098059 (Luconi et ai., 1998).

The cellular targets of activated ERKs include, nuclear-, cytosolic-, cytoskeletal- and

membrane proteins (Bokemeyer et ai., 1996, Gonzales et ai., 1993). In mitotic cells,

the main target of activated ERKs, is the nucleus, where it phosphorylates

transcriptional factors, ultimately resulting in de novo protein synthesis and cell

proliferation. In non-mitotic mature spermatozoa the nucleus plays hardly, if any, a

role in the modulation of their biological function and therefore a nucleur translocation

of ERKs seem unlikely. Thus the presence of ERKs at the level of the equatorial

segment after the AR (Luconi et ai., 1998) might be related to the regulation and/or

activation of proteins that mediate binding and fusion between the sperm and egg

plasma membranes. It has also been demonstrated that a conformational

rearrangement of proteins, which appear to play an important role in the sperm-

oocyte fusion at the level of the equatorial membrane, take place after stimulation of

the AR (Allen & Green, 1995).


108

The downstream targets of ERKs during capacitation and the AR of spermatozoa

remain to be defined. Seeing that both capacitation and AR are earmarked by the

rearrangement of cytoskeletal elements, a possible target might be cytoskeletal

elements such as microtubule-associated proteins (MAP), whose phosphorylation

may be important in the cytoskeletal rearrangements that occur during capacitation

(Duncan & Fraser, 1993).

In conclusion, our data demonstrates that ERKs are directly or indirectly involved in

the acrosome reaction induced by human zona pellucida.

Acknowledgements

This study was supported in part by the Technology and Human Resources for

Industry Programme (THRIP), managed by the National Research Foundation (NRF)

and funded by the Department of Trade and Industry (DTI).


109

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Aitken RJ, Buckingham OW, Harkiss 0 (1996). The extragenomic action of human

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phosphorylation during capacitation. Mol Cell Endocrinol 17:83-93.

Baldi E, Luconi M, Bonaccorsi L, Krausz C, Forti G (1996). Human sperm activation

during capacitation and acrosome reaction: role of calcium, protein

phosphorylation and lipid remodelling pathways. Front Biosci 1:189-205.

Bastiaan H, Franken DR, Wranz P (1999). G-Protein regulation of the solubilised

Human Zona Pellucida-Mediated Acrosome Reaction and Zona Pellucida

Binding. JAssist Reprod Gen 16(6): 332-336.

Berruti G (1994). Biochemical characterisation of the boar sperm 42 kilodalton protein

tyrosine kinase: its potential for tyrosine as well as serine phosphorylation

towards microtubule-associated protein 2 and histone H 2B. Mol Reprod Oev

38:386-392.

Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazon A, Marshall CJ, Parker

PJ, Sugden PH (1994). ET1 and fibroblast growth factors stimulate the MAP-

kinase signalling cascade in cardiac myocytes. The potential role of the

cascade in the integration of the two signalling pathways leading to myocyte

hypertrophy. J Bioi Chem 269(2):1110-1119.

Bokemeyer 0, Sorokin A, Dunn MJ (1996). Multiple intracellular MAP kinase

signalling cascade. Kidney Int 49:1187-1198.


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Breitbart H, Spungin B (1997). The biochemistry of the acrosome reaction. Mol Hum

Reprod 3(3):195-202.

Burgering BM, Bos JL (1995). Regulation of RAS mediated signalling: More than one

way to skin a cat. Trends Biochem Sci 20:18-22.

Burks OJ, Carballada R, Moore HOM, Saling PM (1995). Interaction of a tyrosine

kinase from human sperm with the zona pellucida at fertilization. Science

269:83-86.

Canman CE, Kastan MB (1996). Three paths to stress relief. Nature 384:213-214.

Carr OW, Acott TS (1990). The phosphorylation of a putative sperm micro tubule-

associated protein 2 (MAP2) is uniquely sensitive to regulation. Bioi Reprod

43:795-805.

Cross NL, Morales P, Overstreet JW, Hanson FW (1988). Induction of acrosome

reactions by the human zona pellucida. Bioi Reprod 38:235-244.

Duncan AE, Fraser LR (1993). Cyclic AMP-dependent phosphorylation of epididymal

mouse sperm proteins during capacitation in vitro: identification of an Mr 95 000

phosphotyrosine-containing protein. J Reprod Fertil 97:287-299.

Franken DR, Bastiaan HS, Oehninger SC (2000) Physiological induction of the

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Gonzales FA, Seth A, Raden DL, Bowman OS, Fay FS, Davis RJ (1993). Serum-

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Kopf GS, Visconti PE, Moos J, Galentino-Homer HL, Ning XP (1995). Integration of

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Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, Van Zyl JA,

Smith K (1986). Sperm morphologic features as a prognostic factor in in vitro

fertilization. Fertil Steril 46:1118-1123.

Leyton L, Saling PM (1989). 95 kd sperm proteins bind ZP3 and serve as tyrosine

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Luconi M, Barni, T, Vanelli GB, Krausz C, Marra F, Benedetti PA, Evangelista V,

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Luconi M, Bonaccorsi L, Krausz C, Gervasi G, Forti G, Baldi E (1995). Stimulation of

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modulates tyrosine phosphorylation and tyrosine kinase activity during

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Visconti PE, Bailey JL, Moore GD, Pan 0, Olds-Clarke P, Kopf GS (1995).

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Knobil E, Neil JD (eds), Vol1 2nd ed. New York: Raven Press, 189-317.


"A scientific truth does not triumph by

convincing its opponents and making them

see the light, but rather because its

opponents eventually die and a new

generation grows up that is familiar with it."

- Maxwell Planck -


113

CHAPTER 5

EXTRACELLULAR SIGNAL-REGULATED KINASE ACTIVATION

INVOLVED IN HUMAN SPERM-ZONA PELLUCIDA BINDING

ANDROLOG/A 2002 Vo/34 no. 1pp55-59

SS du Plessis":', C page1 and DR Franken2





Running head: ERK involved in sperm-zona binding

Keywords: spermatozoa, acrosome, ERK, zona pellucida, hemizona

·Corresponding author. SS du Plessis, Department of Medical Physiology and



[email protected]


mailto:[email protected]

114

Summary

In a previous study involving the inhibition of the mitogen-activated protein kinase

(MAPK), extracellular signal regulated kinase (ERK), we found that the very specific

MAPK Kinase (MEK) inhibitor, PD098059, inhibited the zona pellucida (ZP) induced

acrosome reaction. Since an intact acrosome on the spermatozoa is a prerequisite to

ensure tight binding to the ZP, we investigated the zona binding potential of

spermatozoa after PD098059 treatment of sperm followed by exposure to solubilized

human ZP and calcium ionophore (A23187). PD098059 treated spermatozoa

exposed to solubilized ZP bound significantly more to the ZP as compared to control

spermatozoa also exposed to solubilized ZP (26.5±3.7 vs. 13.8±2.8, p<O.05). No

significant differences in binding to the ZP were observed between PD098059

treated and untreated sperm populations after A23187 exposure. These results can

be interpreted to support the idea that the ZP-induced AR is the physiologically

relevant exocytotic event since it is the ZP-induced AR, and not the spontaneous

(culture medium) or A23187 induced AR, which appears to be mediated through an

ERK-mediated signal transduction process.

Introd uction

Before a spermatozoon can penetrate and fertilize the oocyte, it has to undergo

various physiological processes i.e. capacitation (series of membrane and metabolic

transformations), acrosome reaction (AR - fusion of the outer acrosomal membrane

with the plasma membrane leading to exocytosis of enzymes and exposure of new

membrane domains), as well as tight binding to the oocyte (species specific binding

of spermatozoa to the zona pellucida - ZP). The human AR, a Ca2+-dependent

exocytotic event that is regulated by voltage-operated calcium ion channels located


115

in the plasma membrane of the sperm head, is a crucial early step in the fertilization

process, and must be completed prior to fusion with oocytes (Babcock & Pfeiffer,

1987; Florman et aI., 1992; Florman, 1994; Arnouit et aI., 1996; Son et aI., 2000).

Under physiological conditions, sperm voltage operated calcium channels are usually

activated by contact with the ZP (Florman et aI., 1992; Arnouit et aI., 1996), while it

can also be experimentally mediated by progesterone (Foresta et aI., 1993).

The mammalian ZP, and specifically glycoprotein 3 (ZP3), is the main mediator of

sperm-egg recognition, sperm binding and the AR in mammals (Yanagimachi, 1994).

ZP3 induces Ca2+ influx into cytoplasm, leading to increases of intracellular Ca2+ and

pH, and resulting in acrosomal exocytosis (Babcock & Pfeiffer, 1987). The human ZP

has been shown to bind human spermatozoa and to induce the AR of spermatozoa

in both the intact and the solubilized state (Bielfield et aI., 1994; Cross et al., 1988;

Morales et aI., 1989). However, these methods cannot be used routinely in human

studies, due to the scarcity of human material. Instead, several bovine serum albumin

(BSA) neoglyco-proteins were shown to be able to stimulate the AR by interacting

with the putative receptor for ZP3 in human spermatozoa (BrandeIIi et al., 1996;

Blackmore & Eisoldt, 1999).

Extracellular regulated kinase (ERK), a member of the serine/threonine family of

mitogen activated kinases, can be phosphorylated and activated through both G-

protein- and protein tyrosine kinase (PTK) receptor ligand binding in many cell types

(Canman & Kastan, 1996; Page & Doubell, 1996). Both of these receptor types also

play a role in the intracellular signalling systems regulating the processes of

capacitation and AR. In support of this hypothesis, it was previously shown in our


116

laboratories that the ZP-induced AR appears to be mediated through a G-protein-

mediated signal transduction process after functional inactivation of the o-protein

receptor by pertussis toxin (Bastiaan et a/., 1999; Franken et a/., 1996). Aitken et al.

(1996) have also shown that the addition of the tyrosine kinase inhibitor, genistein,

inhibited capacitation, probably by inhibiting the protein tyrosine kinase ZP receptor.

It has recently been shown that ERK's are present in human spermatozoa (Luconi et

a/., 1998). We have also showed recently in our laboratories that the specific

inhibition thereof significantly reduced the ZP stimulated AR in human spermatozoa

(Du Plessis et a/., 2001).

The notion that ERK might play an important regulatory role in sperm ZP binding and

thus sperm-oocyte binding was thus further pursued during this study.


Preparation of sperm sam pies

Semen samples were collected from normozoospermic donors by masturbation after

2-3 days of sexual abstinence. Semen samples were analysed according to the

World Health Organisation criteria (WHO, 1999) together with strict sperm

morphology assessment (Kruger et a/., 1986). Motile sperm fractions were collected

from samples using a slightly modified double-wash swim-up technique. Retrieved

sperm samples were resuspended in synthetic human tubule fluid culture medium

(HTF) (Quin et a/., 1985) supplemented with 3% bovine serum albumin (BSA;

Seravac, Cape Town, South Africa) to a sperm concentration of 10x106 cells/ml.

Before the onset of AR studies, sperm samples were allowed to capacitate at 37°C in


117

5% CO2 for 3 hours in HTF-BSA. Prepared sperm samples were incubated in the

presence or absence of the MEK-inhibitor, PD098059 (P-215, Sigma, Cape Town,

South Africa) at a concentration of 50JlMfor 90 minutes at 37°C.





liquid nitrogen (Hammit et a/., 1991). Twenty four hours prior to each test, oocytes





20 oocytes on the petri dish. A total volume of 5JlI of 10mM HCl was then added to


and controlled. Following solubilization, 5JlI of 10mM NaOH was added to the

solubilized ZP, to render a final zona volume of 10JlI containing 2 ZP/JlI (Liu and

Baker, 1996). The final ZP concentration, after the addition of spermatozoa, was 0.67

ZP/Jll.

Hemizona Assays/Zona Pellucida Binding

Spermatozoa were stimulated with (i) 10JlM A23187 (C-7522, Sigma, Cape Town,

South Africa) for 20 minutes and (ii) 0.67ZP/JlI for 60 minutes in either the presence

or absence of PD098059 (pre-treatment) to elicit the AR (Cross et a/., 1988; Morales


118

et a/., 1989, Franken et a/., 2000) and compared to that of control samples. Control

samples were allowed to spontaneously acrosome react.

Prior to aspiration into Teflon tips during the micro-assay (Franken et a/., 2000), the

sperm/ZP and sperm/A23187 suspensions were gently mixed in a well of a 60 well

micro-titre plate (Microtest plate cat No. P43, Laboratory and Scientific, Cape Town

South Africa). Aspirating HTF droplets into both sides of the Teflon tip sealed off the

sperm suspensions and prevented evaporation from the tip. Each sperm/ZP and

sperm/A23187 suspension was separated from the HTF droplets by air bubbles on

both sides.

Progressive motility of all samples was monitored before and after the incubation

periods. Sperm droplets were carefully placed on separate spots of spotted slides

(MAGV, Germany, XER 201B) and immediately evaluated for percentage live sperm

under inverted phase contrast microscope (Nikon TMS-F, Research Inst.

Johannesburg, South Africa). Only samples with a progressive motility of more than

80% were subsequently used in the experiments.

Both test and control sperm droplets (15!J.1;10x106sperm/ml) were placed on a petri

dish to which hemizonae were added in a match-controlled fashion. Hemizona

assays (HZA) were performed multi-fold and co-incubation was for four hours.

Following the co-incubation period, hemizonae were removed and washed (5x) to

strip the loosely attached spermatozoa from the hemizonae. Hemizonae were then

evaluated, while the number of spermatozoa tightly bound to the ZP was recorded for

each test and matching control. Two individuals using the blind scoring method


119

scored the amount of spermatozoa bound for each hemizona assay. Sperm-zona

binding was also calculated for matched hemizonae assay results and expressed as

a mean percentage or Hemizona Index (HZI) where HZI = Test sperm bound to hemizona/control

sperm bound to hemizona X 100.


Sperm-zona binding results were expressed as the mean number of sperm bound

per hemizona. The Mann-Whitney U test for nonparametric data was used to

compare these binding results. p-values equal or less than 0.05 were considered

statistically significant. A Hemizonae Index was also calculated for matched

hemizonae assay results and expressed as a mean percentage. A HZI of 100%

implies that the compound tested did not interfere with the amount of spermatozoa

that bind to the ZP as compared to that of the control spermatozoa.

Results

Since the acrosome plays an important role during the binding and penetration of the

ZP, the zona-binding capacity of PD098059 treated sperm populations was recorded.

Table 1 illustrates the sperm-zona binding data. Results were obtained after

PD098059 treatment of sperm followed by exposure to solubilized human ZP or

calcium ionophore (A23187). Significantly more of the PD098059 treated

spermatozoa bound to the ZP (mean±SE) as compared to control spermatozoa, after

exposure to solubilized ZP (26.5±3.7 Vs. 13.8±2.8; p<0.05; Table 1). No significant

differences in binding to the ZP were observed between PD098059 treated and

untreated sperm populations after A23187 exposure.


120

Table 1. Sperm-zona binding results after PD098059 treatment followed by exposure

to Calcium lonophore (A23187) and solubilized Zona pellucida (ZP).

Control hemizona assay Test hemizona assay

Spermatozoa treated with different

components alone

Spermatozoa exposed to components

after PD098059 treatment

Component Mean (±SE) no. of

zona-bound

sperm

Component Mean (±SE) no. of

zona-bound

sperm

Culture medium 53.4±10.58 Culture medium 43.3±8.9

(n=15)

A23187

(n=12)

0.67ZP/IlI

(n=12)

13.8±2.8b

(n=12)

A23187

(n=12)

0.67ZP/IlI

(n=12)

22.2±2.534.7±10.2

26.5±3.7C

a Vs. b, p<0.005; b Vs. c, p<0.05

The results of the sperm zona binding of matched hemizonae are expressed as a HZI

in Table 2. It is evident that the MEK-inhibitor, PD098059, did not influence sperm-

zona binding per se as a HZI of 107% was recorded when compared to the binding

potential of control sperm (Table 2. A). A 14% increase in HZI was observed between

PD098059-treated and untreated spermatozoa, after ZP stimulation prior to sperm-

zona binding, when compared to that of their respective control hemizona assays

(Table 2. 0 = 28% Vs Table 2. E = 42%). PD098059 treated sperm populations

exposed to solubilized ZP bound more to each hemizonae as compared to

spermatozoa incubated in a solution containing only ZP (HZI = 240%; Table 2. G).

No real differences could be detected in sperm-zona binding between control and


121

P0098059-treated spermatozoa after A23187 stimulation when compared to that of

their respective control hemizona assays (Table 2. B = 57% Vs. Table 2. C = 54%).

Table 2. Sperm-zona binding results expressed as a Hemizona Index (HZI) after

P0098059 (PO) treatment followed by exposure to Calcium lonophore (A23187) and

solubilized Zona pellucida (ZP).

# Control hemizona assay Test hemizona assay HZI n

A Culture medium PO + Culture medium 107% 5

B Culture medium A23187 57% 5

C Culture medium PO + A23187 54% 5

0 Culture medium ZP 28% 7

E Culture medium PO+ZP 42% 5

F A23187 PO + A23187 107% 4

G ZP PO+ZP 240% 5

H PO + Culture medium PO+ZP 41% 6

I PO + Culture medium PO + A23187 50% 5

(HZI = es IControl X 100)

Discussion

The present results illustrate the involvement of extracellular regulated kinase in the

ZP stimulated AR (or physiological relevant exocytotic event) as opposed to the non-

physiological A23187 induced event. The increase in binding of P0098059 pre-

treated spermatozoa to the oocyte (13.8±2.8 Vs. 26.5±3.7 and HZI 28% Vs. 42%) is

proof that the ZP induced AR was indeed inhibited and underline the possible

regulatory effect of P0098059 and its subcellular targets on the ZP-induced AR.


122

PD098059 is a specific inhibitor of the activation of the ERK kinase (MEK), the kinase

upstream of ERK, responsible for the dual phosphorylation and activation of ERK

following G-protein coupled- and protein tyrosine kinase receptor ligation and

activation, both in vitro and in vivo (Alessi et ai., 1995). Aitken et al. (1996) showed

that the addition of genistein, a tyrosine kinase inhibitor, inhibited capacitation,

probably by inhibiting the protein tyrosine kinase ZP receptor, while the functional

inactivation of the Gi-protein receptor by pertussis toxin inhibited the ZP-induced AR

which appear to be mediated through a G-protein mediated signal transduction

process (Bastiaan et ai., 1999). Luconi et al. (1998) also showed that in vitro

capacitation stimulates a sustained and concomitant increase in tyrosine

phosphorylation and kinase activity of ERK; this activation of ERK was strongly

inhibited when capacitation was performed in the presence of the MAPK cascade

inhibitor PD098059 (Luconi et al., 1998).

PD098059 also has effects seemingly unrelated to the inhibition of ERK, as shown by

its functional inactivation of voltage-dependent calcium (Ca2+) channels that

participate in fertilization of the marine worm, Urechis caupo (Gould & Stephano,

2000). Voltage operated calcium channels can be classified into low voltage-

activated and high voltage-activated calcium channels on the basis of thresholds for

activation. High voltage-activated Ca2+ channels having high thresholds can be

further classified by their pharmacological properties into L-, N-, P/Q-, and R-types

(Birnbaurner et ai., 1994; Dunlap et ai., 1995). Widely used high voltage operated

calcium channel antagonists can selectively block these channels. Stimulation of

sperm with ZP depolarises the sperm membrane potential. ZP (specifically ZP3)

stimulation activates a depolarisation mechanism with the characteristics of a poorly


123

selective cation channel. Pre-treatment of sperm with PD098059 possibly prevents

activation of the Ca2+ -selective channel by ZP3/zonae pellucidae as is the case with

pertussis toxin treatment (Florman et ai., 1995). The results can be interpreted to

support the idea that the ZP-induced AR is the physiologically relevant exocytotic

event since it is the ZP-induced AR, and not the spontaneous (culture medium) or

A23187 induced AR, which appears to be mediated through a ERK-mediated signal

transduction process.

Although it is generally accepted that the spermatozoa must be acrosome-reacted to

complete penetration of the ZP (Franken et al., 1991), the exact site of the AR has

not been defined and appears to differ between species. PD098059 treatment of

human spermatozoa does not affect the ability of spermatozoa to bind to structural

intact human ZP. The results indicate the importance of intact acrosomes on the

spermatozoa to ensure tight binding to the ZP, i.e., those sperm populations with a

decreased AR; namely, the PD098059 treated spermatozoa bound significantly

higher numbers of sperm during HZA conditions (Du Plessis et al., In press).

Acknowledgements

This study was supported in part by the Technology and Human Resources for

Industry Programme (THRIP), managed by the National Research Foundation (NRF)

and funded by the Department of Trade and Industry (DTI).


124

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Luconi M, Barni T, Vanelli GB, Krausz C, Marra F, Benedetti PA, Evangelista V,

Francavilla S, Properzi G, Forti G, Baldi, E (1998) Extracellular regulated signaI-

kinases modulate capacitation of human spermatozoa. Bioi Reprod 58: 1476-

1489.

Morales P, Cross NL, Overstreet JW, Hanson FW (1989) Acrosome-intact and


Bioi 133: 385-392.


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Page C, Doubell AF (1996) Mitogen-activated protein kinase (MAPK) in cardiac

tissue. Mol Cell Bio 157:49-57.

Quin P, Kerin JF, Warnes GM (1985) Improved pregnancy rate in human in vitro

fertilization with the use of a medium base on the composition of human tubal


Son W-Y, Lee J-H, Han C-H (2000) Acrosome recation of human spermatozoa is

mainly mediated by alfa1H type calcium channels. Mol Hum Reprad 10, 893-

897.




33.

Yanagimachi R (1994) Mammalian Fertilization. In: The Physiology of Reproduction.

Vo11, 2nd ed. Knobil E, Neil JD (eds). Raven Press, New York, pp 189-317.


"Every great advance in science has

issued from a new audacity of

imagination. "

- Joh n Dewey -


128

CHAPTER 6

Phosphatidylinositol 3-kinase inhibition enhances human sperm

motility and sperm-zona pellucida binding.

Archives of Andrology 2002, submitted

SS du Plessis 1,·, DR Franken2, M Luconf and E Baldi3





3Dipartimenti di Fisiopatologia Clinica, Unita di Andrologia, Universitá di Firenzi, viale

Pieraccini 6, 1-50139,Firenzi, Italy

Ruqning head: PI3-Ks effect on sperm motility and binding

Keywords: spermatozoa, acrosome, PI3-kinase, zona pellucida, hemizona

'Corresponotnq author. SS du Plessis, Department of Medical Physiology and



[email protected],.


mailto:[email protected],.

129

Summary

Various signalling pathways are involved in the regulation of sperm motility,

capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an

important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility,

however no studies as of yet has been done to determine the effect of this inhibitor

on other sperm parameters. Here we investigated the role of PI3K on human sperm

motility, acrosome reaction and sperm-oocyte binding by using the specific PI3K

inhibitor LY294002. We demonstrate that in vitro incubation of washed unselected

spermatozoa with LY294002, increased the percentage motility and progressive

motility in asthenozoospermia patients as evaluated by computer-aided sperm

analysis. The compound furthermore did not influence the acrosome reaction, whilst

it further did enhance sperm-oocyte binding. Our results therefore imply that PI3K

negatively affect sperm motility and oocyte binding. We subsequently suggest a

possible therapeutic role for PI3K inhibitors in the treatment regime for

asthenozoospermia.

Introd uction

Spermatozoon prerequisites for initiation of fertilization include motility, capacitation

and acrosome reaction. Normal motility patterns are needed to deliver the sperm to

the site of fertilization, while concurrently it also has to undergo a series of functional

biochemical and biophysical modifications named capacitation in order to render the

ejaculated spermatozoa competent for fertilization of the oocyte. Finally a timeous

acrosome reaction, which is an exocytotic process physiologically, induced by ligand

(ZP3)-receptor interaction, ultimately lead to sperm-zona binding.


130

Many patients who attend infertility clinics are diagnosed with asthenozoospermia

that result in impaired fertilization rates in vivo as well as during in vitro procedures

(Oehninger, 2001). Even with the onset of advanced assisted reproductive

techniques like in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI),

the quest to increase the fertilizing ability of semen samples is a very relevant and

ongoing process. It is expected that simplified and more cost-efficient therapeutic

modalities will be developed as additional basic (cellular-molecular) and clinical

knowledge is gained (Edirisinghe et a/., 1995; Oehninger, 2001). It is therefore one of

the goals of reproduction physiologists to try and increase the motility of spermatozoa

among asthenozoospermic patients without interfering with the normal physiological

processes needed for fertilization.

Various studies have demonstrated that several pharmacological agents such as

SpermSolute (based on a proteinase - trypsin) and pentoxifylline can definitely

improve sperm motility (Tesarik et a/., 1992; Krausz et a/., 1994; Lanzafame et a/.,

1994; Minhas & Ripps, 1996; Figenschau & Bertheussen, 1999; Terriou et a/., 2000).

However, the possibility of using such agents is limited due to the presence of non-

responding subjects (Krausz et a/., 1994) and the possible toxic effects of these

molecules (Centola et a/., 1995)

Even though it is known that phosphatidylinositol 3-kinase (PI3K) is present in mouse

spermatozoa (Feng et a/., 1998), it was only recently demonstrated that LY294002,

the specific inhibitor of PI3K, increased the progressive motility of spermatozoa in

humans (Luconi et a/., 2001). Dolci et al. (2001) reported that stem cell factor (SCF)

acts as a mitogenic factor in cultured c-kit-expressing spermatogonia and that both


131

mitogen-activated protein kinase kinase (MEK)- and Pl3K-dependent pathways are

required for the proliferative response. The mitogenic effect is not accompanied by an

increase in total cellular amount of cyclin D3, but it is associated with a rapid change

in its subcellular localisation. It was also shown that SCF is an anti-apoptotic factor

for spermatogonia, but the MEK- or the Pl3K-dependent pathways are not sufficient

on their own to promote the survival response. It was further suggested that the

binding of c-kit to SCF in mature sperm cells may result in the activation of PLCy1

and PI3K, leading to receptor autophosphorylation and ultimately may playa role in

capacitation and/or the acrosome reaction (Feng et al., 1998).

PI3K's are SH2 domain-containing proteins, which is known to be important in

phosphoinositide-mediated intracellular signalling pathways in numerous cell types.

And is heterodymeric, consisting of a p85 regulatory (adapter) subunit and a p110

catalytic subunit. It phosphorylates the 3'-OH group of the inositol ring in inositol

phospholipids and the most important of these is thought to be the conversion of

phosphatidylinositol (Ptdlns)-4,5-bisphosphate to Ptdlns-3,4,5-trisphosphate (Fisher

et al., 1998). PI3K's have been identified in most cells studied. It has been implicated

in the priming of a series of signalling cascades involved in the regulation of many

processes of somatic cells including mitogenesis, differentiation, motility, cell survival,

oocyte maturation, cell chemotaxis, membrane ruffling, DNA synthesis, receptor

internalisation, vesicular trafficking and metabolic control (KapelIer & Cantley, 1994;

Vanhaesebroeck et ai., 1997; Wymann & Pirola, 1998; Anderson et ai., 1999). It has

also been suggested that PI3K might possibly be involved in the AR elicited by the

mannose-BSA agonist by making use of the unrelated PI3K inhibitor, wortmannin

(Fischer et al., 1998).


132

As of yet no studies have been performed to test the effect of this inhibitor on the ZP

induced AR as well as sperm-zona binding potential. The study aimed to investigate

the role of PI3Ks in human sperm motility, AR and sperm-zona binding by using the

PI3K inhibitor, LY294002.


Preparation of sperm samples

Semen samples were collected from volunteers (average age = 25.81±1.09 years;

n=18) and analysed and classified according to the WHO criteria (WHO, 1999).

Only normozoospermic samples were used to perform AR and sperm-oocyte binding

tests. Motile sperm fractions were collected after a double wash in synthetic human

tubal fluid culture medium (HTF) (Quin et a/., 1985) and 60-minute swim-up

technique in HTF supplemented with 3% bovine serum albumin (BSA; Seravae, Cape

Town, South Africa). Retrieved sperm samples were resuspended in synthetic HTF-

BSA to a sperm concentration of 1Ox106 cells/ml. These fractions were then

capacitated in the presence or absence of the PI3K-inhibitor, LY294002 (LY; 101JM;

120min; 37°C, 5% CO2 and 95% humidity).

The semen samples used to perform computer-aided sperm analysis (CASA)

underwent a double wash in HTF. The pellet containing the spermatozoa was then

resuspended to its original volume in HTF + BSA. After preparation these samples


133

were incubated (37°C, 5% CO2 and 95% humidity) for 2h in the presence or absence

of the PI3K antagonist, lY294002 (10j.lM; 120min).


Oocytes were retrieved from post mortem derived ovarian material. Approval was

obtained from the Institutional Review Board and fully complies with the legal and

ethical guidelines of the South African Medical Research Council. Retrieved oocytes

were stored in a dimethylsulfoxide/sucrose solution at -196°C in liquid nitrogen

(Hammit et al., 1991). Twenty four hours prior to each test, oocytes were removed

from storage and thawed at 37°C. Retrieved oocytes were placed in 0.25M sucrose

and 3% BSA in HTF. On the day of the experiment, 20 oocytes were placed in a drop

on a petri dish, after which the HTF was removed under microscopic vision (Olympus

SZ40; Wirsam Scientific, Cape Town, South Africa), leaving only the 20 oocytes on

the petri dish. A total volume of 5f..l1of 10mM HCl was then added to the oocytes on

the petri dish; solubilization of the ZP was microscopically observed and controlled.

Following solubilization, 5f..l1of 10mM NaOH was added to the solubilized ZP, to

render a final zona volume of 10f..l1containing 2 ZP/f..ll(Du Plessis, 2001). The final ZP

concentration, after the addition of spermatozoa, was 0.67 ZP/f..ll.

Sperm kinematics

The samples prepared for motility studies were analysed using a Hamilton-Thorne

(Hamilton-Thorne Research, Beverly, MA) IVOS analyser (integrated visual optical

system) with standard set-up parameters. Washed spermatozoa were resuspended

in a capacitation medium containing HTF with 3% BSA. Computer assisted semen

analysis (CASA) was performed on each sample at 3 hours after collection using a


134

10.0!J.mchamber (Makler) and a sampling rate of 30 frames at 60Hz. A minimum of

100 cells and four fields was analysed for each aliquot. All analyses were performed

at 37°C.

Acrosome reaction studies

Acrosomal statuses of spermatozoa stimulated with 0.67ZP/!J.1for 30 minutes in

either the presence or absence of LY294002 (pre-treatment) were determined

according to procedures published elsewhere (Cross et al., 1988; Morales et ai.,

1989, Franken et ai., 2000) and compared to that of control samples. These

stimulation procedures were performed as micro-assays as previously described

(Franken et ai., 2000).

Spermatozoa from the different experiments were fixed and air-dried, after which the


(FITC-PSA; 125!J.g/ml;L-0770; Sigma, Cape Town, South Africa). Two individuals

scored a minimum of 200 spermatozoa blindly for each different sample. The

following staining patterns were evaluated as acrosome reacted spermatozoa: (i)

patchy staining on acrosomal region, (ii) distinct staining in the equatorial region

occurring as an equatorial bar and (iii) no staining over the entire sperm surface.


Both LY294002 pre-treated (test) and control sperm samples (15!J.1droplets;

1Ox106sperm/ml) were placed on a petri dish to which hemizonae were added in a

match-controlled fashion. Duplicate hemizona assays (HZA) were performed and

sperm-zona binding was assessed after four hours of co-incubation. Following the


135

co-incubation period, hemizonae were removed and washed (5x) to strip the loosely

attached spermatozoa. Hemizonae were then evaluated, while the number of

spermatozoa tightly bound to the ZP was recorded for each test and matching

control. Experiments where less than 20 spermatozoa bound to a control hemizona

were discarded. Sperm-zona binding was expressed as a percentage or Hemizona

Index (HZI) where HZI - Test sperm bound to hemizonal X 100 A HZI of- Control sperm bound to hemizona .

100% would therefore imply that the compound tested did not interfere with the

amount of spermatozoa that bind to the ZP as compared to that of the control

spermatozoa.


Sperm kinematics results, percentage acrosome reacted sperm and numbers of

sperm bound to the hemizonae are expressed as mean±SE. Student's t-test for

paired data was used to compare the results of the motility, acrosome and hemizona

studies. Pearson's test was used for correlation tests when comparing different

motility parameters. P-values equal or less than 0.05 were considered statistically

significant.

Results

Sperm kinematics

Overall semen samples (n=18) showed a statistically significant increase in

progressive motility after co-incubation with the PI3K inhibitor, LY294002,

(31.88±3.75% vs. 37.31±3.74%, P=0.020). A complete shift in velocity distribution

also occurred. According to CASA more of the static, slow and medium cells became


136

activated and the rapid population increased from 43.63±4.80% in the control

samples to 50.38±4.20% in the LY294002 pre-treated samples (P=0.025). The rest of

the sperm kinematics parameters did not change statistically significantly after

LY294002 treatment in the donor population as a whole.

The results were subsequently divided into two groups (Table 1) based on the %

motility of each donor's sperm sample. The groups were classified as >50% motility

(normozoospermic) and <50% motility (asthenozoospermic) according to WHO

(1999) criteria. The effect of PI3K inhibition is much more pronounced in the

asthenozoospermic group compared to the normozoospermic group. The percentage

motile and percentage progressive motile spermatozoa increased statistically

significantly by 22% (from 29.25% to 51.25%; P=0.009) and 10.25% (from 15.00% to

25.25%; P=0.008) respectively after LY294002 treatment (Table 1). The velocity

distribution results of this group also showed a statistically significant change with the

rapid moving population increasing by 15.25% (from 21.50% to 36.75%; P=0.010)

and the static population decreasing by 22% (from 70.75% to 48.75%; P=0.009).

An inverse effect occurred in the amplitude of lateral head displacement (ALH)

between the two groups after LY294002 treatment. (The normozoospermic

populations ALH increased from 4.23±0.17lJm to 4.44±0.14IJm (P=0.046) while the

asthenozoospermic populations decreased from 5.00±0.43IJm to 4.38±0.30IJm

(P=0.017)). When a" of the control and LY pre-treated CASA results were pooled and

analysed together a positive correlation (r=0.6487; P<0.0001; ~=0.4208; n=36) was

found between the progressive motility values and the beat cross frequency (BCF)

values (Figure 1). No correlation existed between progressive motility and ALH, while


137

upon comparing BeF to ALH (Figure 2) a negative correlation was found (r=-O.3462;

P=O.026; ~=O.1199) in this group.

Table 1. Sperm kinematics results of all the samples as well as when divided into

normozoospermic and asthenozoospermic donors in the presence and absence of

LY294002

All samples >50% Motility <50% Motility P

Control LY Control LY Control LY(n=18) (n=18) (n=12) (n=12) (n=6) (n=6)

Motile 62.87±6.14 69.06±4.69 74.08±3.62 75.00±4.38 29.25±1O.44 51.25±9.38 * 0.009(%) * *Progressive 31.88±3.75 37.31±3.74 38.50±3.38 41.33±4.01 15.00±5.48 25.25±6.26 * 0.020(%) * * # # # 0.008

Path velocity 64.23±2.86 65.31±2.50 65.70±2.87 68.23±2.36 59.78±7.99 56.58±5.49(VAP, um/s)

Progressive velocity 54.85±3.09 55.54±2.59 56.71±2.90 58.39±2.28 49.25±9.14 47.00±6.75(VSL, urn/s)

Track speed 100.89±4.07 105.09±2.88 101.36±4.84 106.84±3 .28 99.48±8.54 99.83±5.91(VCL, um/s)

Lateral amplitude 4.43±0.18 4.43±0.12 4.23±0.17 4.44±0.14 5.00±0.43 4.38±0.30 * 0.046(ALH, urn) * * # # #0.017

Beat frequency 26.20±0.97 27.01±1.08 27.17±1.09 27.08±1.25 23.48±1.64 26.80±2.43(BCF, Hz)

Straightness 82.56±146 82.81±1.31 83.50±0.99 83.50±1.01 79.75±5.31 80.75±4.60(STR, %)

Linearity 54. 13±1.96 53.31±1.97 55.83±1.54 55.00±1.50 49.00±6.23 48.25±6.52(LIN, %)

Elongation 69.44±0.94 68.00±0.63 68.33±0.51 67.67±0.75 72.75±3.12 69.00±1.08(%)

Area 4.35±0.15 4.16±0.12 4.42±0.16 4.16±0.14 4.15±0.41 4.18±0.25(urn sq)

Rapid 43.63±4.80 50.38±4.20 51.00±4.11 54.92±4.28 21.50±7.51 36.75±8.26 * 0.025(%) * * # # # 0.010

Static 37.13±6.14 30.88±4.68 25.92±3.61 24.92±4.35 70.75±10.44 48.75±9.38 * 0.009(%) * *


138

35-N 30:r;-u,0 2510

200

• • •• •- -----+ - .• •

• •10 20 30 40 50 60 70

PM (%)

Figure 1. Correlation between beat cross frequency (BCF) and progressive motility

(PM) of pooled experiments (n=36). (r=0.6487; P<0.0001; 1=0.4208)

35 ~--------------------------------~-N 30:r;-

•• • •• •_.-+- .~- _.....~-• •••

••• •

u,o 2510

203 4 5

ALH (J.lm)

6 7

Figure 2. Correlation between beat cross frequency (BCF) and amplitude of lateral

head displacement (ALH) of pooled experiments (n=36). (-r=0.3462; P=0.026;

1=0.199)


139

Acrosome reaction

Due to the scarcity of human ZP, the effect of LY294002 on the spontaneous AR or

the ZP-induced physiologically relevant exocytotic event was only determined in a

limited number of normozoospermic sperm samples. ZP stimulation increased the

%AR by a statistically significant 106.1% (P=0.017) in the control sample (from

10.18±1.924% to 20.98±1.805%), while a statistically significant increase of 112.3%

was recorded in the LY pre-treated group (from 8.525±2.234 to 18.1±1.882). Though

decreases in AR were detected in spontaneous (less 16.3%) and ZP induced AR

(less 13.7%) after LY pre-treatment, it was not of significant value. It can thus be

deducted that exposure of spermatozoa to the PI3K inhibitor, LY294002, did not

influence the spontaneous or ZP induced acrosome reactions during these

experiments (n=5; Figure 3).


140

25

20

0::: 15«:::e 100

5

0C ZP LY ZP+LY

Figure 3. Histogram showing the percentage acrosome reaction (mean±SE) of

control (C) spermatozoa and spermatozoa after exposure to zona pellucida (ZP),

PI3K antagonist LY294002 (LY) or both ZP and LY294002 JZP+L Y). LY did not inhibit

or increase the spontaneous or ZP induced acrosome reactions (n=5). IC vs. ZP

(P=O.017); C vs. LY (P=O.60B); C vs. ZP+L Y (P=O.007), ZP vs. LY (P=O.009); ZP vs.

ZP+LY (P=O.323); LVvs. ZP+L Y (P=O.019)]


141

Sperm-zona pellucida binding

A statistically significant difference (P=0.024) could be detected between the number

of LY294002 pre-treated spermatozoa (49.72±2.7; range 29.0-80.0 and median =49.5) and control spermatozoa (45.11±2.6; range 28.0-70.0 and median = 44.0) that

bind to the hemizonae's respectively (Figure 4). The ratio between LY pre-treated

(test) and untreated (control) samples produced a HZI of 112% ± 5%. This suggests

a trend that more LY pre-treated spermatozoa did bind to the hemizona.

ca 600 *N 500... 40caE... 30CJ)c.f/) 20....0 10.0Z 0

Control Test

Figure 4. Histogram showing the number of control and LY294002 pre-treated (Test)

spermatozoa (mean±SE) tightly bound to each hemizona respectively (n=18). (*

P=0.024 vs. control)

Discussion

Controversies do exist regarding sperm motility enhancers and their effects on sperm

viability and fertilizing ability. In the present study it was demonstrated that the

addition of LY294002, a very specific inhibitor of phosphatidylinositol 3-kinase, is able


142to increase the motility and progressive motility of human spermatozoa. These results

are strongly supported by similar studies performed by luconi et al. (2001). We

specifically show that the addition of l Y294002, to double washed sperm samples,

increase progressive motility and the percentage rapid cells in our general donor

population. In addition we demonstrate that our results are even more exaggerated in

the asthenozoospermic population. Not only did the progressive motility and rapid cell

population increase dramatically, but also the percentage motile cells increased while

percentage static cells decreased markedly. luconi et al. (2001) also showed

increases in Vel, VAP and VSl (and other motion parameters) as measured by

eASA. We however did not find any significant increases in these parameters but did

see increasing trends in these parameters among all our sperm populations. Not

finding statistic significance in this study regarding the above mentioned parameters

can be ascribe to making use of smaller sample populations as well as differences in

preparational techniques (e.g. centrifuged, washed, unselected sperm vs. swim-up

selected sperm). The molecular mechanisms underlying the stimulatory effects of

l Y294002 on sperm motility are still poorly understood. The process of development

and maintaining of motility in mammalian spermatozoa is rather complex and

involves the integration of and crosstalk of several signalling pathways, including

adenylate cyclase/cAMP/PKA, calcium and phosphorylation/dephosphorylation of

proteins (Tash and Bracho, 1994). luconi et a/., (2001) speculate that in

spermatozoa PI3K might be involve in phosphorylating or dephosphorylating proteins

involved in motility or in the generation of reactive oxygen species (ROS) which has a

negative impact on motility.

It can also be deducted from our results that BeF is closely related to progressive

motility and thereby an increase in BeF might lead to an increase in progressive


143motility. It was also shown that BCF and ALH correlates negatively. It can thus be

further deducted that in order for BCF to increase, there are less time for the sperm

head to be displaced sideways and thereby decreasing ALH.

Knowing that the PI3K signalling pathway is involved in eliciting of the AR (Fisher et

a/., 1998), it was therefore important to investigate the effects of LYon this reaction.

In our hands LY294002 did not affect the spontaneous or the ZP induced AR even

though a slight decline in AR was observed in all 5 experiments between the ZP and

the LY+ZP groups. Furthermore no significant (P=0.163) difference in AR was

observed between progesterone stimulated spermatozoa with or without LY

treatment (results not shown). Luconi (personal communication) also found that LY

exerts no effect on both the spontaneous and progesterone stimulated AR, thereby

supporting the current data. Contrary to our findings, Fischer et al. (1998) showed a

decrease in the AR elicited by the mannose-BSA agonist by making use of the

unrelated PI3K inhibitor, wortmannin. Yet wortmannin was found not to inhibit the

acrosome reaction induced by either A23187 or progesterone (Fischer et ai., 1998).

This may imply that the cellular pathways involved in bringing about the acrosome

reaction induced by the different agonists (ZP3, Progesterone, Mannose-BSA,

A23187) do not involve PI3K to the same extend, or perhaps that the need for PI3K

in the pathway is somehow bypassed. It may also imply that LY294002 and

wortmannin have different mechanisms of action or differ in their specificity to inhibit

PI3K. In this regard wortmannin has indeed been shown to inhibit other enzymatic

activities (Cross et a/., 1995) including MAPK and extracellular signal regulated

kinases (ERK) at doses from 250nM (Ferby et ai., 1996). The presence of ERK has

been demonstrated in human spermatozoa (Luconi et al., 1998) and implicated to be

involved in the ZP-induced AR (Du Plessis et ai., 2001). It can therefore be


144speculated that the decrease in AR in wortmannin treated samples after exposure to

the mannose-BSA agonist may not only be due to PI3K inhibition, but also due to the

possible simultaneous inhibition of ERK. However as half-maximal inhibition of MAPK

activation occurs at 300nM this must be ruled out as Fisher et al. (1998) used the

inhibitor at concentrations ranging between 10nM and 100nM.

When considering that the different agonists (i.e. ZP3, Progesterone, Mannose-BSA)

all stimulate different signalling pathways through binding to different membrane

receptors, it is obvious that it will result in different cellular responses. From the

literature it is evident that various crosstalk mechanisms are involved in PI3K and

RAS/RAF/ERK signalling. It is known that RAS can both activate RAF/ERK as well as

PI3K (Sears & Nevins, 2002; Krasilnikov, 2000). PI3K on the other hand can also

activate the RAS/RAF/ERK pathway through mitogen signalling (Krasilnikov, 2000)

(see Figure 5). Taking into consideration that mannose-BSA stimulation elicit the AR

through both Gi-protein receptor binding (BrandeIIi et al., 1996) and receptor tyrosine

kinase binding (Fisher et al., 1998), and that PI3K is regulated by receptor tyrosine

kinase activity, it can explain why PI3K inhibition can inhibit the mannose-BSA

induced AR (Fisher et ai., 1998). Wortmannin will inhibit PI3K, thereby also inhibiting

the PI3K activated RAS/RAF/ERK pathway needed for eliciting of the AR (Du Plessis,

2001). There is no evidence in the literature that the mannose-BSA tyrosine kinase

receptor is directly linked to the RAS/RAF/ERK pathway, but only through PI3K. In

our experiments we stimulated the AR with solubilized ZP (ZP3), thereby eliciting the

AR through both a Gi-protein receptor and ZRK binding which directly activates

RAS/RAF/ERK and PI3K through RAS (see Figure 5). Inhibition of PI3K by

LY294002 thus only inhibited the PI3K pathway and not the direct RAS/RAF/ERK

pathway activated independent of PI3K signalling or activation through protein kinase


145C (PKC). The activation of RAS by ZP binding to ZRK can also stimulate PI3K,

thereby enhancing a possible positive feedback on ERK activation (e.g. via PKC) as

well as other mediators in the process of AR signalling (see Figure 5).

A statistically significant increase in sperm-oocyte binding after LY treatment was

also detected. It was also previously shown in our laboratory that the frequency of

sperm/zona pellucida collision rates has an influence on zona binding (OzgOr &

Franken, 1996). This increase in binding can therefore be ascribed to the fact that

more spermatozoa are motile and progressively motile, thereby increasing the

chances of them colliding with the oocyte. Myles and Primakoff (personal

communication) showed similar results in the mouse model by finding that incubation

of mouse sperm and oocyte in the presence of LY significantly increases sperm-zona

binding.

Compared with signalling pathways in many types of somatic cells, the signal

transduction pathways of the mammalian acrosome reaction (pellucida glycoprotein)

are still very poorly understood (Fisher et al., 1998). One of the chief reasons for this

is that the sperm receptor(s) that bind to zona protein 3 (ZP3) and physiologically

induce the acrosome reaction remain to be conclusively identified (Brewis & Moore,

1997). Indeed, in the human, about ten sperm determinants in spermatozoa have

been reported to be involved (listed and referenced in Benoff, 1997). The most likely

scenario is that several sperm determinants are involved in zona binding and function

in concert as part of a complex (Kopf et al., 1995; Brewis & Moore, 1997).

Our results imply that PI3K negatively regulates sperm motility and increase sperm-

zona binding without influencing the acrosome reaction. This suggests that


146LY294002 can be used as a tool to enhance the motility of sperm samples during

preparation for IVF from patients with low sperm motility, thereby ultimately opening a

new prospective for severe oligoasthenozoospermic males to enter IVF rather than

ICSI programs.


147

Mannose-BSA

TKR Plasma membrane

\\\\\\\\\\\\\\\\\\

LY294002Wortmann in

Figure 5. Possible interactions between the different signal transduction pathways

invoked during the acrosome reaction. (ZP3 = zona pellucida glycoprotein 3; ZRK =Zona Receptor Kinase; TKR = mannose tyrosine kinase receptor; dashed lines =hypothesised activation of ERK; G;-coupled receptor pathway not shown)


148

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The erection is the last gasp of modern

manhood. If men can't continue to

produce erections, they're going to evolve

themselves right out of the human

.species.

- Time Magazine, 5 May 1998-


153CHAPTER 7

The effect of acute in vivo sildenafil citrate (VIAGRA™) treatment on

semen parameters and sperm function

Monduzzi Editore 2002, In Press

SS du Plessis 1,*, PS de Jongh 1 and DR Franken2





Running head: Effect of sildenafil citrate on sperm parameters and function

Keywords: sildenafil citrate, spermatozoa, acrosome, zona pellucida, motility

*Corresponding author. SS du Plessis, Department of Medical Physiology and



[email protected],.


mailto:[email protected],.

154

Summary

Viagra TM is a powerful treatment for male erectile dysfunction of various aetiologies.

Sildenafil citrate, the active ingredient of Viagra TM, is a specific and potent inhibitor of

phosphodiesterase type 5 and thus enhances the activity of the nitric oxide-cGMP

pathway that promotes penile erection due to cGMP accumulation. In this study we

investigated whether acute in vivo sildenafil citrate (50mg orally) administration

modifies seminal parameters, induction of the acrosome reaction, sperm-oocyte

binding and sperm motility. No changes in the macro- and microscopical seminal

parameters were caused by sildenafil citrate when compared to placebo. Sildenafil

citrate also did not initiate or potentiate the acrosome reaction. Sperm-oocyte

binding, smooth path velocity, straight line velocity and the percentage rapid cells all

increased after sildenafil citrate treatment. These results suggest a clinical usefulness

for sildenafil citrate in the enhancing of fertilizing ability of inherently poor quality

sperm or for treatment during assisted reproductive techniques.

Introduction

Erectile dysfunction (ED) is a widespread condition that has a negative impact on

quality of life, affecting both older and younger men in an estimated 10% of the adult

population (Feldman et ai., 1994). Until recently no effective oral therapy existed and

available treatments were highly cumbersome or invasive (Purvis et aI., 2002).

Sildenafil citrate (ViagraTM, Pfizer), a cyclic nucleotide phosphodiesterase (PDE)-

inhibitor, was the first oral agent to be successfully introduced for the management of

ED in which there is no established organic cause (Boolell et ai., 1996b). When

administered before sexual activity, it produces reliable efficacy, good tolerability and

rapid absorption that yields prompt onset of action (Boolell et al., 1996a).


155Cyclic nucleotide phosphodiesterases regulate intracellular levels of cyclic 3',5'

adenosine monophosphate (cAMP) and cyclic 3',5' guanosine monophosphate

(cGMP) by hydrolysing them to the corresponding 5' monophosphates (Conti et aI.,

1995). Cyclic nucleotides serve as second messengers for a wide variety of

extracellular signals such as nitric oxide, neurotransmitters, hormones, light and

odourants. As of yet nine different POE iso-enzymes (POE1 to POE9) have been

described and found to be present at various concentrations in human tissues

(Soderling et aI., 1998a; Fabbri et aI., 1999).

Nitric oxide synthase, a cGMP inducer (Lewis et aI., 1996), and two distinct POE

isoforms (POE1 and POE4) are present in human sperm cells (Fisch et aI., 1998). It

was also shown in previous studies that mRNA coding for cAMP-specific POE

(POE4A) isoforms are present in mature rat and mouse germ cells (Naro et al., 1996)

and that the expression of these isoforms is maximal in round spermatids and is

maintained in mature spermatozoa (Soderling et aI., 1998b).

Phosphodiesterase type-5 (POE5) is the predominant POE iso-enzyme for the

degradation of cGMP in the corpus cavernosum. Sildenafil citrate (Viagra™) is a

specific and potent inhibitor of this cGMP-specific POE5 enzyme and thus enhances

the activity of the nitric oxide-cGMP pathway (Figure 1) that promotes penile erection

due to cGMP accumulation (Ballard et al., 1998; Glossman et al., 1999). It also has

minor inhibitory effects on POE6 and POE1 activities (ICsD= 0.034 and 0.281Jmol/l

respectively) (Morales et al., 1998). Sildenafil citrate mimics cGMP and interacts with

the catalytic site of POE. Ouring normal erections sexual stimulation lead to an

increase in nitric oxide production which in turn stimulate cGMP production. These

increased cGMP levels causes trabecular smooth muscle relaxation, cavernosal


156artery dilatation, increased cavernosal pressure and penile erection. Sildenafil citrate

potently enhances the relaxant effect of nitric oxide on the human corpus

cavernosum by increasing the value of cGMP in this tissue. However sexual

stimulation still remain mandatory in order to produce its pharmacological effect,

since it has no direct relaxant effect on the corpus cavernosum (Fabbri et ai., 1997;

Ballard et ai., 1998; Goldstein et al., 1998). Viagra TM is rapidly absorbed and acts

within 30 minutes and reach maximum plasma concentrations within approximately 1

hour. The absolute bioavailability is 41% due to first-pass metabolism and is rapidly

cleared from the body with a plasma elimination half-life of 3-4 hours (Nichols et ai.,

2002).

It is likely that men with ED will use sildenafil citrate as an aid to procreation,

especially by younger patients who have ED e.g. secondary to spinal cord injury. It is

also known that a degree of ED may be present in the male infertile partner

especially when assisted reproductive techniques are necessary. These men have

difficulties in producing spermatozoa on demand at the time of egg fertilization. Tur-

Kaspa et al. (1999) showed that these forms of temporary erectile dysfunction can be

successfully treated with sildenafil citrate. In this context however, the local affects of

sildenafil citrate on sperm function have not been researched extensively.

A large number of drugs can be transported into the seminal fluid, where they have

direct effects on function, physiology, metabolism or genetic composition of

spermatozoa (Pichini et ai., 1994). Several drugs have been shown to affect sperm

motility in particular; these include compounds with PDE-inhibitory activity that have

the potential to increase motility (Schoenfeld et a/., 1975; Turner et ai., 1978). One

such drug, pentoxifylline, which is a cAMP PDE inhibitor, has been shown to


157stimulate human sperm motility both in vitro and in vivo (Shen et aI., 1991). It has

also been demonstrated that human sperm cells contain as yet uncharacterised POE

isoforms which are different from POE1 and POE4, and that the in vitro inhibition of

sperm POE1 and POE4 iso-enzymes by specific inhibitors stimulates acrosome

reaction (AR) and sperm motility (Fisch et al., 1998). It is also known that after acute

oral administration of 100mg-sildenafil citrate, the drug reaches a concentration of

0.1-0.3 ,",mol/I in the ejaculate (Pfizer, Viagra™ data sheet). This concentration is

consistent with possible inhibitory actions of sildenafil citrate on sperm POE isoforms

(Fabbri et aI., 1999).

Sildenafil citrate is rapidly becoming the treatment of choice for EO of various

aetiologies even though many of its potential effects are still being investigated

(Edwards and Muirhead, 2002). The drug is relatively lipophilic (log07.4=2.7) and

would therefore be expected to distribute into the seminal fluid (Purvis et aI., 2002)

where it subsequently might have important local effects/actions which still remain

unknown. We therefore conducted a study to investigate and evaluate the effects of

an acute single oral dose of sildenafil citrate (50mg) on a number of seminal, sperm

functional and morphology parameters in young healthy male volunteers.


Penile/sexual

stimulation

NANC&endothelial

cells

158

Corpus cavernosummooth muscle cell

PenileerectionRELAX

Figure 1. Enhancement of the NO-cGMP mechanism of penile erection by si/denafil

citrate. Inhibition by sildenafil citrate results in elevation of cGMP concentrations in

the corpus cavernosum, which induces relaxation of corpus cavernosal smooth

muscle, vasodilatation, increased blood flow to the penis, increased intracavernosal

pressure and penile erection. NO = nitric oxide; cGMP = cyclic guanosine

monophosphate; POE5 = phosphodiesterase type 5; NANG = noradrenergic-

noncholinergic neurones; GTP = guanosine triphosphate.


159


Study design and subjects

The study design consisted of a prospective double blind, placebo-controlled,

crossover, two period clinical investigation. The study blind was broken after

completion of all investigations. All subjects enrolled spontaneously in the study after

giving an informed consent to the study protocol, which was approved by the Ethical

Committee of our institution. The inclusion criteria included normal erectile function,

normal electrocardiogram, no prior or concomitant serious illness or consumption of

medications during the 3-month period prior to the study. In a double-blind fashion,

subjects randomly received one of the two treatments, that is SOmg sildenafil citrate

(Viagra™, Pfizer, Sandton, RSA) or placebo to ingest orally. All subjects were asked

not to ejaculate for a minimum of 3 days before each dose of study drug. They were

not permitted to consume products containing alcohol, caffeine or methylxanthines or

cigarette smoking for a period of three days before each session of the study. The

subjects were also required not to use prescription or over-the-counter drugs during

the course of the study. All study medications were taken under supervision with

SOmlof water. The washout period consisted of a 7-day period in which no

medication was given. After this time, all subjects were crossed over to receive the

alternative treatment.

Written informed consent was obtained from each subject before study entry.

Subjects who met the inclusion criteria underwent a pre-study screening in the 3

weeks before the start of the study that consisted of a physical examination,


160measurement of supine blood pressure and pulse rate, a 12-lead electrocardiogram

and a urine test.

An in vitro study was also conducted in parallel in order to investigate the effects of

increased levels of exogenous cGMP on sperm motility, AR and sperm-oocyte

binding. 8-bromo-cGMP (8-Br-cGMP), a non-hydrolyzable cGMP analogue, was used

to increase intracellular cGMP levels in vitro, as it is lipid soluble and therefore able to

enter the cell quite easily (in contrast to cGMP that cannot penetrate the cell

membrane). The drug dissolved easily in water and was used at a final concentration

of 20IJM(60minutes).

Semen processing I Preparation of sperm samples

Semen samples were collected 1 hour after ingestion of the test drug by means of

masturbation according to the World Health Organisation guidelines (WHO, 1999).

Motile sperm fractions were collected from samples using a slightly modified double-

wash swim-up technique. Retrieved sperm samples were resuspended in synthetic

human tubal fluid culture medium (HTF) (Quin et ai., 1985) supplemented with 3%

bovine serum albumin (BSA; Seravac, Cape Town, South Africa) to a sperm

concentration of 1Ox106 cells/ml. Before the onset of AR and sperm-oocyte binding

studies, sperm samples were allowed to capacitate at 37°C in 5% CO2 for 3 hours in

HTF-BSA. Excess spermatozoa that were not used for motility or AR studies were

cryopreserved (using SpermFreeze; FertiPro N.v, Beernem, Belgium) in 0.5 ml

straws using a slow-freeze method (Mahedevan and Trounson, 1993) in order to

perform matched hemizona assays at a later stage.


161Macro and microscopical seminal parameters

Ejaculate volume was measured volumetrically and pH was determined with Multistix

Reagent Strips (Bayer Corporation, Tarrytown, NY, USA). Liquefication, viscosity and

appearance of each sample were judged visually. Sperm concentration, sperm count

and sperm head size (area) was acquired by means of computer assisted sperm

analysis (CASA, see later). An assessment of morphology was made after staining

with eosin Y and Harris haemotoxylin and spermatozoa was evaluated according to

World Health Organisation guidelines (WHO, 1999) together with strict sperm

morphology criteria (Kruger et a/., 1986).

Motility studies

Sperm kinematics was assessed after a 3D-minute liquefication period at room

temperature by means of Computer assisted semen analysis (CASA). The analysis

was performed on an Integrated Visual Optical System for sperm analysis (IVOS,

Hamilton Thorne Research, Beverly, MA) with standard set-up parameters. The raw

semen was diluted 1:1 in HTF-BSA and placed in a disposable fixed-depth slide.

(20.0Ilm, Makler) at an image capture rate of a 30 frames at 60Hz. A minimum of 200

cells and three fields was analysed for each aliquot. All analyses were performed at

3rc. The following measurements were performed:

Counts:

Total, Motile, Progressive

% Motile, % Progressively Motile

Rapid, Medium, Slow and Static Cells

Concentrations

Total, Motile, Progressive (millions/ml)

Rapid, Medium, Slow and static Cells (millions/ml)


162Mean Values

VAP: Smoothed Path Velocity (microns/sec)

VCL: Track Velocity (microns/sec)

VSL: Straight Line Velocity (microns/sec)

AlH: Amplitude of lateral Head Displacement (microns)

BCF: Beat Cross Frequency (hertz)

LIN: Linearity (ratio of VSLlVCl)

STR: Straightness (ratio of VSLlVAP)

Area: Head size (square microns)





liquid nitrogen (Hammit et al., 1991). Twenty four hours prior to each test, oocytes





20 oocytes on the petri dish. A total volume of 5JlI of 10mM HCl was then added to


and controlled. Following solubilization, 5JlI of 10mM NaOH was added to the

solubilized ZP, to render a final zona volume of 10JlIcontaining 2 ZP/Jll. The final ZP

concentration, after the addition of spermatozoa, was 0.67 ZP/Jll.


163Acrosome reaction studies

Spermatozoa from both the Viagra or placebo groups were challenged with (i) 10llM

calcium ionophore A23187 (C-7522, Sigma, Cape Town, South Africa) for 30

minutes, (ii) 1lJg/ml progesterone for 30 minutes, (iii) 0.67ZP/1l1for 30 minutes or (iv)

left to spontaneously elicit the AR. The acrosomal status of these spermatozoa were

subsequently determined according to procedures published elsewhere (Cross et al.,

1988; Morales et al., 1989, Franken et al., 2000).

Spermatozoa from the different experiments were fixed and air-dried, after which the


(FITC-PSA; 125Ilg/ml; L-0770; Sigma, Cape Town, South Africa). Two individuals

scored a minimum of 200 spermatozoa blindly for each different sample. The

following staining patterns were evaluated as acrosome reacted spermatozoa: (i)

patchy staining on acrosomal region, (ii) distinct staining in the equatorial region

occurring as an equatorial bar and (iii) no staining over the entire sperm surface.


Straws containing the cryopreserved spermatozoa were thawed rapidly at 3rC for

10 minutes. Cryoprotectant was removed with the dropwise addition of P1 medium

followed by centrifugation at 600 9 for 10 minutes and resuspension in 0.5ml fresh

medium (Mahedevan & Trounson, 1993). Both test (sildenafil citrate) and control

(placebo) sperm droplets (15111;10x106sperm/ml) were placed on a petri dish to

which hemizonae were added in a match-controlled fashion. Hemizona assays (HZA)

were performed in duplicate and co-incubation was for four hours. Following the co-

incubation period, hemizonae were removed and washed (5x) to strip the loosely

attached spermatozoa from the hemizonae. Hemizonae were then evaluated by two


164individuals using the blind scoring method, while the number of spermatozoa tightly

bound to the ZP was recorded for each test and matching control. Sperm-zona

binding was also expressed as a percentage or a Hemizona Index (HZI) where the

HZI - Test sperm bound to hemizona/ 100 A HZI f 100°/ ould- Control sperm bound to hemizona X. 0 10 W

therefore imply that the compound tested did not interfere with the amount of

spermatozoa that bind to the ZP as compared to that of the control spermatozoa.


All results are expressed as the mean ± the standard error. Data were compared

using Student's t-test for paired data. P-values equal or less than 0.05 were

considered statistically significant. All statistical analysis were performed using

GraphPad PRISM version 2.01.

Results

Demographic characteristics, safety and tolerability

Twenty healthy male volunteers were enrolled in the study. The subjects were all

Caucasian and between 20 and 33 years of age (23.182 ± 0.689). No significant

medical conditions relevant to the study were noted before entry or during the study.

Adverse events considered by the investigator to be related to treatment occurred in

6 of 20 subjects (30%) following administration of sildenafil citrate and in 1 of 20

subjects (5%) following administration of placebo (Table I). The most frequently

reported adverse events were flushing, headache and abnormal vision (blue vision)

and were generally mild to moderate in severity. No subject discontinued treatment

due to adverse effects related to sildenafil citrate.


165Table I: Incidence of adverse events following treatment with placebo or sildenafil

citrate (n=20).

Placebo Sildenafil 50mg

Treatment related adverse events 1/20 6/20

Flushing 0 3

Headache 0 2

Abnormal vision 0 2

Spontaneous erections 1 1

Macro- and microscopical seminal parameters

Table II shows the macroscopical seminal parameters after acute in vivo sildenafil

citrate or placebo administration. Liquification, appearance and viscosity of the

semen samples from both groups were normal as according to the WHO criteria.

Mean values of semen volume (2.533±0.320ml vs. 2.373±0.313ml) and pH

(8.182±0.076 vs. 8.318±0.076) did not show significant variations between the two

treatment groups.

Table II: Initial macroscopic appearance and evaluation of semen after either

placebo or sildenafil citrate administration (n=20).

Semen parameters Placebo Sildenafil 50mg

Liquefication <60 minutes <60 minutes

Appearance Normal Normal

Viscosity Normal Normal

Ejaculate Volume (ml) 2.533 ± 0.320 2.373 ± 0.313

pH 8.182 ± 0.076 8.318 ± 0.076

p

0.432

0.096


166

Similarly sildenafil citrate was equivalent to placebo in its effects on all microscopical

secondary semen analysis parameters, including sperm count, sperm concentration

and morphology (Table III). All these variables were also within the normal ranges as

predefined by the study centre. The area of the sperm head size (square microns), as

determined by CASA, was not affected by sildenafil citrate.

Table III: Effects of acute in vivo administration of sildenafil citrate on microscopical

secondary semen analysis parameters (n=20).

Semen parameters Normal Placebo Sildenafil P

values 50mg

Sperm count (x106) 100-500 220.8 ± 45.892 232.2 ± 54.453 0.449

Sperm concentration (x106/ml) 50-150 73.72 ± 13.32 77.52 ± 13.29 0.627

Morphology (% normal forms) >15 15.059 ± 0.961 14.765 ± 0.893 0.281

Area (urn sq) 3.400 ± 0.228 3.350 ± 0.394 0.872

Acrosome reaction

From Table IV it is evident that the addition of exogenous cGMP did not affect the AR

between treatment groups (control vs. 8-Br-cGMP) when left to spontaneously

undergo the AR or when stimulated with A23187 or progesterone. The AR is thus

independent of treatment with 8-Br-cGMP at a concentration of 20!JM.

The results of the various AR studies from the clinical trial are displayed in Figure 2.

When left to spontaneously undergo the AR, the placebo group showed a 13.94 ±


1672.542% AR while the sildenafil citrate group showed no statistical significant

difference (14.06 ± 2.128%; P = 0.753) in AR. It can be seen that after calcium

ionophore (A23187) stimulation the percentage AR in both groups was statistically

significantly elevated (Placebo: 23.37 ± 2.831%, P=0.0002; Sildenafil: 26.37 ±

3.598%, P=0.0001). Progesterone stimulation had a similar effect and also increased

acrosome reactions in both groups with significant margins to above that of the

spontaneous levels (Placebo: 21.21 ± 3.306%, P=0.004; Sildenafil: 24.90 ± 3.788%,

P=0.007). Exposure to solubilized zona pellucida also increased the acrosome

reactions significantly to above that of spontaneous levels in both groups (Placebo:

25.64 ± 5.797%, P=0.026; Sildenafil: 26.17 ± 3.898%, P = 0.014). The acrosome

reactions between treatment groups (placebo vs. sildenafil) did not differ significantly

after A23187 (P=0.893), progesterone (P=0.728) and zona pellucida (P=0.957)

stimulation and were thus independent of treatment.

Table IV: The effect of in vitro intracellular cGMP elevation by the addition of 8-8r-

cGMP (20fJM) on eliciting of the acrosome reaction (n=6).

Stimuli Control 8-Br-cGMP P

Spontaneous 16.500±3.085 17.167±3.911 0.444

A23187 23.667±3.273 23.000±6.611 0.287

Progesterone 21.333±3.896 20.857±2.852 0.225


168

35c.2 3013i 25~ 20

i 15ob 10ct:-;;e. 5

1"miii;,l" Placebo_ Sildenafil

n

s A p ZP

Figure 2: The effects of double-blind placebo or 50mg-sildenafil citrate administration

on the sperm acrosome reaction (n=20).

S = spontaneous, A = A23187, P = progesterone, ZP = zona pellucida.

Placebo: S vs. A = 0.0002; S vs. P = 0.004; S vs. ZP = 0.026

Sildenafi/: S vs. A = 0.0001; S vs. P = 0.0073; S vs. ZP = 0.0147

Sperm-zona pellucida binding

Incubation of spermatozoa in the presence of 8-Br-cGMP lead to increased sperm-

zona binding and a HZI of 134% was determined (n=6, in duplicate). This percentage

indicates that more spermatozoa bound to their respective matched hemizonaes after

exposure to 8-Br-cGMP (20IJM).

The number of spermatozoa that bound to each matched hemizona (Figure 3) was

not statistically significantly influenced by acute in vivo sildenafil citrate administration

even though a hemizona index of 148.75% was recorded. These results suggest that

a nearly 49% higher binding rate occurred after acute sildenafil citrate ingestion.


169

" 40c::::so.c 30Elo.Cl)C.fI) 20....olo.

.! 10E::::sZ

Sildenafil

Figure 3: The effect of double-blind placebo or 50mg-sildenafil citrate administration

on the average number of spermatozoa tightlylfirmly bound to each hemizona (n=10;

P=O.281).

Sperm kinematics results

On average more than 800 cells (817.8±71.92), but not less than 200 cells were

evaluated in order to determine the various sperm motility parameters. The mean

values for the sperm motility parameters were all within the normal ranges as

predefined by the study centre (Table V). No significant difference was observed

between placebo and sildenafil citrate for percentage motile, percentage progressive

motile and percentage static cells. Vel, AlH, BeF, STR and LIN also did not differ

between treatments. It was however found that both VAP (58.61±2.258IJm.s-1 vs.

64.11±2.195IJm.s-\ P=0.047) and VSl (47.28±2.250IJm.s-1vs. 51.92±2.249IJm.s-1,

P=0.046) increased statistically significantly after sildenafil citrate treatment.

Furthermore it was found that after sildenafil citrate treatment a shift occurred from

the medium, slow and static cell populations towards the rapid cell population thereby


170increasing the percentage rapid cells significantly (44.47±4.782% vs. 50.88±5.180,

P=0.009).

The percentage motile (61.0 ±3.488 vs. 82.00±2.415; P=0.036; n=6) percentage

progressive motile (27.33±2.926 vs. 35.50±4.180; P=0.026; n=6) and percentage

rapid (35.00±3.479 vs. 45.67±4.344; P=0.014; n=6) spermatozoa also increased

significantly after incubation of spermatozoa in the presence of 8-Br-cGMP (201JM,

60minutes).


171

Table V: Effects of 8-Br-cGMP (in vitro) and sildenafil (in vivo) on different sperm motility parameters as measured by CASA.

Parameter Normal Control 8-Br-cGMP P Placebo Sildenafil Pvalues {n=6} {n=6} {n=20} {n=20}

Motile >50 61.00 ± 3.488 82.00 ± 2.451 0.037 67.39 ± 5.892 68.22 ± 5.673 0.850(%)Progressive motility >15 27.33 ± 2.927 35.50 ± 4.180 0.027 30.89 ± 3.809 33.00 ± 3.801 0.530(%)Smoothed Path Velocity 54.96 ± 3.256 58.58 ± 4.898 0.190 58.61 ± 2.528 64.11 ± 2.195 0.047(VAP - um/s)Straight Line Velocity >25 49.28 ± 3.261 51.93 ± 4.869 0.265 47.28 ± 2.250 51.92 ± 2.249 0.046(VSl - um/s)Track Velocity 72.38 ± 4.325 75.98 ± 4.606 0.288 90.18 ± 3.407 94.64 ± 4.009 0.324(VCl - urn/s)Amplitude of lateral Head 3.10 ± 0.202 3.00 ± 0.089 0.542 3.929 ± 0.1350 4.194 ± 0.2190 0.202Displacement (AlH - urn)Beat Cross Frequency 24.12 ± 1.492 24.03 ± 0.674 0.949 24.11 ± 0.8050 24.59 ± 1.136 0.656(BCF - Hz)Straightness 86.50 ± 1.440 85.67 ± 1.291 0.497 77.20 ± 1.377 78.88 ± 1.252 0.447(STR - %)linearity 64.83 ± 2.518 65.67 ± 2.620 0.662 53.61 ± 1.684 54.61 ± 1.589 0.621(LIN - %)Rapid >25 35.00 ± 3.479 45.67 ± 4.344 0.014 44.47 ± 4.782 50.88 ± 5.180 0.009(%)Static <50 32.33 ± 7.298 22.667 ± 6.547 0.272 37.24 ± 6.822 32.35 ± 7.014 0.239(%)


172

Discussion

A total of 20 male volunteers were enrolled in this prospective double blind, placebo-

controlled, crossover, two period clinical investigation to determine the

pharmacodynamic effects of sildenafil citrate on sperm function and ejaculate quality.

Sildenafil citrate was well tolerated by the volunteers, producing no serious adverse

events or clinically important changes in vital signs. The most frequently reported

adverse events were generally consistent with those reported in other studies (Purvis

et a/., 2002; Boolell et a/., 1996; Goldstein et a/., 1998; Morales et a/., 1998).

In this study a comparison of acute sildenafil citrate treatment vs. placebo showed no

effect on a wide variety of macroscopical seminal measures including liquefication,

appearance, viscosity, ejaculate volume or pH. Sperm count, sperm concentration,

morphology and area of sperm head size was also not influenced. These results are

in line with those reported in similar studies (Aversa et a/., 2000).

It was shown that exogenous cGMP did not initiate or potentiate the AR of

capacitated spermatozoa, yet it is well known from the literature that cGMP serve as

a signal transducer mediating the AR. When this signalling pathway is activated by

using 8-Br-cGMP, effects are only documented at concentrations of higher than

0.5mM with peak activity at 1mM (Rotem et a/., 1998; Revelli et a/., 2001). This is

most likely due to PKA stimulation because cGMP can interact with the regulatory

subunit of PKA when used at concentrations higher than 100IJM(Lincoln & Cornwell,

1993). In our experiments we used a concentration of 201JM,which did not result in

the initiation of the AR. Capacitated spermatozoa also underwent an AR when


173

challenged with calcium ionophore, progesterone and zona pellucida plus in

combination with the POE inhibitor sildenafil citrate, but not with sildenafil citrate

alone. These data suggest that sildenafil citrate (50mg orally) by itself cannot initiate

the AR nor can it potentiate the AR of capacitated spermatozoa, as it most probably

cannot elevate intracellular cGMP levels high enough through its inhibition of POE5.

Furthermore, Lefievre et al. (2000) similarly showed that POE inhibitors by

themselves had no effect on the AR.

Fisch et al. (1998) showed by means of immunocytochemical staining that different

types of POE are located at different sites in the spermatozoon e.g. POE4 localises

mainly to the mid-piece, while POE1 is found more prominently in the sperm head.

These observations support the idea that differential POE localisation within the

spermatozoon can allow for selective modulation of sperm function through the

regulation of distinct pools of cAMP (Fisch et al., 1998).

Sildenafil citrate is a controversial member of a drug class (POE inhibitors) previously

shown to either have some effects on sperm motility (Schoenfeld et al., 1975; Turner

et al., 1978; Lefiévre et al., 2000) or no effects on motility at all (Purvis et al., 2000;

Aversa et al., 2000).

In this study a comparison of sildenafil citrate vs. placebo on a wide variety of

kinematics parameters showed no effect on percentage sperm motility, percentage

progressive motility, track velocity, amplitude of lateral head displacement, beat cross

frequency, straightness, linearity or percentage static cells. Borderline statistical

significant changes were however observed in smoothed path velocity and straight-


174

line velocity after sildenafil citrate administration. The most significant change of note

was observed in the increase in percentage rapid cells after sildenafil citrate

administration. Elevation of intracellular cGMP levels by 8-Br-cGMP also increased

percentage motile, percentage progressively motile and percentage rapid cells

significantly. Both Cuadra et al. (2000) and lefievre et al. (2000) showed that the

inhibition of POES through the addition of sildenafil citrate also enhanced certain

motility parameters. Cuadra et al. (2000) found an increase in sperm progressive

motility and hyperactivation, while lefievre et al. (2000) showed increases in VCl,

AlH and hyperactivation.

The hemizona indexes of 148 % after sildenafil citrate and 134% after 8-Br-cGMP

administration refer to increases in sperm-oocyte binding. These increases in binding

can possibly be explained by the fact that, combined with various increases in

different motion characteristics, more spermatozoa are rapidly motile thereby

increasing the chances of them colliding with the oocyte. This theory fits in with

results previously obtained in our laboratory, that the frequency of sperm/zona

pellucida collision rates has an influence on zona binding (OzgOr& Franken, 1996).

This increase in hemizona index is further proof that sildenafil citrate, and therefore

POE inhibition, does not elicit the AR as only AR intact cells can bind to the oocyte.

lefiévre et al. (2000) discovered that the inhibition of sperm POE by sildenafil citrate

was associated with a significant increase in cAMP and that sperm POE activity

measured with cGMP as substrate was threefold lower than when measured with

cAMP. There was also a dose-dependent increase in cAMP levels in spermatozoa

incubated with sildenafil citrate (lefiévre et ai., 2000). It was demonstrated that


175

spermatozoa contain POE that can hydrolyse both cAMP and cGMP (Lefiévre et aI.,

2000). However, whereas cAMP has often been linked to sperm function, the role of

cGMP in sperm functions remains still unclear. The intracellular mechanism

responsible for the sildenafil-induced cAMP increase in human spermatozoa is

unknown, but it can be hypothesised that sildenafil citrate could act on POEs other

than type 5 (Lefiévre et al., 2000). Sildenafil citrate induced similar changes in sperm

motility parameters as nonselective POE inhibitors such as pentoxifylline (Calogero et

aI., 1998; Nassar et aI., 1999) caffeine (Cheng & Boettcher, 1981; Leclerc et aI.,

1996) and IBMX (Leclerc et aI., 1996). These observations also suggest that

sildenafil citrate probably acts on types 1 and 4 POE because cAMP has been

implicated in the regulation of sperm motility (Calogero et al., 1998) through

activation of PKA (Yanagimachi, 1994). It was also shown by Lefiévre et al. (2000)

that sildenafil citrate increased capacitation and the associated tyrosine

phosphorylation of p105/81, 2 fibrous sheath proteins. These 2 latter processes are

recognised to be cAMP-dependent (Leclerc et aI., 1996).

In conclusion, inhibition of POE by sildenafil citrate produces an increase in sperm

kinematics parameters as well as sperm-oocyte binding, but no macroscopical or

microscopical changes occurred. It also suggests that POE is not strongly involved in

the events leading to the human sperm AR.

Although the incidence of ED increases with age, younger men are also affected

(Feldman et aI., 1994). As such, it is very likely that a significant number of patients

will use sildenafil citrate as an aid to procreation. When studying erectile dysfunction

drugs the focus was always on obtaining sexual satisfaction and never about sperm


176

quality. The results of this study clearly indicate that single oral doses of sildenafil

citrate (50mg) can be safely administered without concern on the part of the

physician or the patient regarding adverse effects on sperm or ejaculate quality.

Furthermore, the ability of sildenafil citrate to enhance sperm kinematics results and

sperm-oocyte binding without affecting the AR may be clinically useful. These

findings could have important implications in the use of sildenafil citrate in assisted

reproductive technology to enhance fertilizing potential of inherently poor quality

sperm with less invasive treatments such as intrauterine inseminations, thereby

avoiding more involved procedures such as IVF or ICSI.


177

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Calogero AE, Fishel S, Hall J, Ferrara E, Vicari E, Green S, Hunter A, Burrello N,

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(1996). Nitric oxide synthase and nitrite production in human spermatozoa:

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Mahedevan M, Trounson A (1993). Effect of cryoprotective media and dilution

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spermatozoa by caffeine. Fertil SteriI26:158-161.

Shen MR, Chiang PH, Yang RC, Hong CY Chen SS (1991) Pentoxifylline stimulates

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Knobil E, Neil JD (eds), Vol1 2nd ed. New York: Raven Press, 189-317.


"Today, if you are not confused, you are

just not thinking clearly."

- U. Peter-


182

CHAPTER 8

CONCLUSIONS AND RECOMMENDATIONS

8.1 Conclusion

The physiological acrosome reaction occurs upon interaction of the spermatozoon

with the zona pellucida protein ZP3 and is of fundamental importance in the

fertilization of the oocyte by the spermatozoon. This is followed by the liberation of

several acrosomal enzymes and other constituents that facilitate penetration of the

zona and expose molecules on the sperm equatorial segment that allow fusion of the

sperm membrane with the oolemma. The molecular mechanisms and the signal

transduction pathways mediating the processes of motility, capacitation and the

acrosome reaction have been partially defined and appear to involve modifications of

intracellular calcium and other ions, lipid transfer and phospholipid remodelling in the

sperm plasma membrane as well as changes in protein phoshorylation. However

there are still many unanswered questions regarding human sperm interaction with

the oocyte.

8.1.1 Motility

From the results obtained in this study it was evident that:

i) Phosphatidylinositol 3-kinase negatively regulates sperm motility.

ii) Inhibition of phosphodiesterase type 5 by sildenafil citrate, and therefore a possible

accumulation of cGMP in the spermatozoon, lead to an increase in certain sperm

kinematics parameters.


183

The process of development and maintaining of motility in mammalian spermatozoa

is rather complex and involves the integration and crosstalk of several signalling

pathways, including adenylate cyclase/cAMP/PKA, calcium and

phosphorylation/dephosphorylation of various substrate proteins (Tash and Bracho,

1994).

The molecular mechanisms underlying the stimulatory effects of the PI3K inhibitor,

LY294002, on sperm motility are still poorly understood. At this stage we can only

speculate that in spermatozoa, PI3K might be involved in the phosphorylating or

dephosphorylating of proteins involved in motility. Another possibility is that PI3K is

involved in the generation of reactive oxygen species (ROS), which has a negative

impact on motility (Luconi et ai., 2001).

The increase in motility due to in vivo Viagra TM treatment is a novel finding and only

speculations can be made regarding the intracellular mechanisms underlying the

finding at this stage. Sildenafil citrate induced similar changes in sperm motility

parameters as non-selective POE inhibitors such as pentoxifylline (Calogero et al.,

1998; Nassar et al., 1999), caffeine (Cheng & Boettcher, 1981; Leclerc et al., 1996)

and IBMX (Leclerc et ai., 1996). The intracellular mechanism responsible for the in

vitro sildenafil citrate-induced cAMP increase in human spermatozoa is also

unknown, but it can be hypothesised that sildenafil citrate could act on POEs other

than type 5 (Lefiévre et al., 2000). These observations also suggest that sildenafil

citrate probably acts on types 1 and 4 POEs because cAMP has been implicated in

the regulation of sperm motility (Calogero et al., 1998) through activation of PKA

(Yanagimachi, 1994).


184

Both of these motility-enhancing effects need to be investigated further in order to

clarify their exact mechanisms of action.

8.1.2 Acrosome reaction

From the results obtained in this study it was evident that:

i) Extracellular signal regulated kinases are directly or indirectly involved in the signal

transduction pathway through which the zona pellucida-induced acrosome reaction

(physiologically relevant exocytotic event) is mediated and not the spontaneous or

calcium ionophore induced acrosome reaction.

ii) Phosphatidylinositol 3-kinase increase sperm-zona binding without influencing the

acrosome reaction.

iii) Inhibition of phosphodiesterase type 5 by sildenafil citrate and therefore possible

accumulation of cGMP produce an increase in sperm-oocyte binding. It also suggests

that phosphodiesterase type 5 is not strongly involved in the events leading to the

human sperm acrosome reaction.

By combining these results with current facts from the literature, a hypothesised

schematised representation of the intracellular signal transduction pathways involved

in the zona pe/lucida-induced acrosome reaction will be shown in Figure 1 and

discussed subsequently.

The ZP3 glycoprotein binds to at least two receptors in the plasma membrane. One

receptor is a Gj-coupled receptor that activates phospholipase C (PLC) r11. The other

receptor is a tyrosine kinase receptor (TKR) coupled to PLCy• Binding to the Gj-


185

coupled receptor would activate adenylate cyclase (AC) leading to the elevation of

cAMP and PKA activity. PKA activates a voltage-dependent Ca2+channel in the outer

acrosomal membrane, which releases Ca2+from the interior of the acrosome to the

cytosol. This relative small rise in [Ca2+]jcould result in the activation of PLCy.The

products of phosphatidyl-inositol biphosphate (PIP2) hydrolysis by PLCB1and PLCy,

diacylglycerol (DAG) and inositoltrisphosphate (IP3) lead to PKC translocation to the

plasma membrane and its subsequent activation. This increase in [Ca2+]j can be

mimicked by the addition of a calcium ionophore (e.g. A23187), which will also result

in the activation of PLCy, and PKC activity. PKC opens a voltage-dependent Ca2+

channel in the plasma membrane, increasing the [Ca2+]jeven more. PKC activation

also results in the activation of ERK, through its ability to phosphorylate and activate

an upstream mediator of the ERK cascade, Raf. Raf will activate and phosphorylate

the ERK kinase, MEK, which in turn activates ERK. The Gj-coupled receptor and

TKR can also activate a Na+/H+exchanger, leading to alkalisation (pH increase) of

the cytosol. The increase in [Ca2+]j and pH will lead to membrane fusion and

acrosomal exocytosis.

From the literature it is evident that various crosstalk mechanisms are involved in

phosphatidylinositol 3-kinase (PI3K) and RAS/RAF/ERK signalling. It is known that

RAS can both activate RAF/ERK as well as PI3K (Sears & Nevins, 2002; Krasilnikov,

2000). PI3K on the other hand can also activate the RAS/RAF/ERK pathway through

mitogen signalling (Krasilnikov, 2000). In our experiments we stimulated the AR with

solubilized ZP (ZP3), thereby eliciting the AR through both a Gj-protein receptor and

ZRK binding which directly activates RAS/RAF/ERK and PI3K through RAS.

Inhibition of PI3K by LY294002 thus only inhibited the PI3K pathway and not the


186

direct RAS/RAF/ERK pathway activated independent of PI3K signalling or activation

through PKC. The activation of RAS by ZP binding to ZRK can also stimulate PI3K,

thereby enhancing a possible positive feedback on ERK activation (e.g. via PKC) as

well as other mediators in the process of AR signalling.

It is well known from the literature that cGMP serve as a signal transducer mediating

the AR. When this signalling pathway is activated by using 8-Br-cGMP, effects are

only documented at concentrations of higher than 0.5mM with peak activity at 1mM

(Rotem et al., 1998; Revelli et al., 2001). This is most likely due to PKA stimulation

because cGMP can interact with the regulatory subunit of PKA when used at

concentrations higher than 100IJM(Lincoln and Cornwell, 1993).

Compared with signalling pathways in many types of somatic cells, the signal

transduction pathways of the human acrosome reaction (pellucida glycoprotein) are

still very poorly understood (Fisher et aL, 1998). One of the chief reasons for this is

that the sperm receptor(s) that bind to zona protein 3 (ZP3) and physiologically

induce the acrosome reaction remain to be conclusively identified (Brewis and

Moore, 1997). Indeed, in the human, about ten sperm determinants in spermatozoa

have been reported to be involved. The most likely scenario is that several sperm

determinants are involved in sperm-zona binding and acrosome reaction, functioning

in concert as part of a complex, while crosstalk can also occur between different

intracellular signalling systems (Kopf et aI., 1995; Brewis and Moore, 1997). [In

Fisher et al., 1998]


187

A thorough understanding of signal transduction in human spermatozoa will

ultimately yield information regarding the nature of receptors to which these signal

transduction pathways are coupled as well as the intracellular effectors that ultimately

regulate sperm function. Moreover, an understanding of these regulatory pathways

will be essential for the future development of clinical approaches designed to

enhance or preclude fertilization.

ZP3

PDES

1

Plasma membrane

Ca'+

[cGMPj > O.SmM -------

Outer acrosomal membrane

Figure 1: Hypothesised signal transduction pathways and possible interactions

between them. (Dashed lines = hypothesised activation/stimulation)


188

8.2 Recommendations

In the field of reproductive biology the emphasis are for various reasons currently

shifting away from the female and directed more towards finding solutions for male

contraception and male infertility.

Due to the fact that hormonal manipulation (as is the basis of most female

contraceptives) as a male contraceptive has so far not been very successful as it can

lead to various side effects and even render the male permanently infertile, the need

has arisen to look at other fronts. On the other hand, the cure for male infertility factor

due to asthenozoospermia often leads to expensive intracytoplasmic sperm injection

(IeSI) procedures which is still surrounded by controversy regarding the outcome of

the health of the conceptus (Oehninger, 2001; Ola et al., 2001; Simpson & Lamb,

2001). If the possibility exists to increase the motility of spermatozoa without

interfering with the rest of the sperms normal physiological processes needed for

fertilization, these patients can automatically become candidates for relatively less

expensive IVF treatments (Edirisinghe et aI., 1995).

With this in mind the following recommendations can be made regarding solutions at

the level of intracellular signalling in the spermatozoon:

i) It is recommended that in the quest to preclude fertilization or finding a male

contraceptive the possible role of ERK inhibitors and MAPK agonists and antagonists

need be further investigated. Our data demonstrate that ERKs are directly or

indirectly involved in the acrosome reaction induced by human zona pellucida. The

results also indicate the importance of intact acrosomes to ensure tight binding to the


189

ZP, while it is generally accepted that the spermatozoa must be acrosome-reacted to

complete penetration of the zona pellucida.

ii) Our results imply that PI3K negatively regulates sperm motility and increase

sperm-zona binding without influencing the acrosome reaction. We subsequently

suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for

asthenozoospermia. This suggests that LY294002 can be used as a tool to enhance

the motility of sperm samples during preparation for IVF from patients with low sperm

motility, thereby ultimately opening a new prospective for severe

oligoasthenozoospermic males to enter IVF rather than ICSI programs.

iii) The ability of sildenafil citrate to enhance sperm kinematics results and sperm-

oocyte binding without affecting the acrosome reaction may be clinically useful.

These findings could have important implications in the use of sildenafil citrate in

assisted reproductive techniques in order to enhance the fertilizing potential of

inherently poor quality sperm with less invasive treatments such as intra-uterine

inseminations, thereby avoiding more involved procedures such as IVF or ICSI.

8.3 Future research

The requirement for sperm capacitation as well as the acrosomal status of the

spermatozoa travelling through the cumulus oophorus needs to be studied further.

Although efforts have been made to establish the characteristics of the proteins

present in the sperm surface that acts as the receptor for ZP3 glycoprotein, the exact

function for these proteins in the mechanism associated to the ZP3-induced AR has

not yet been elucidated. Thus future studies should focus on determining the exact


190

nature of these receptors, their structure and function. The presence of neuronal

receptors (such as GABA-like and glycine receptor) in mammalian spermatozoa with

a role in AR, either induced by progesterone or solubilized ZP needs also be

investigated (Morales and Llanos, 1996). In addition, the AR, a major regulatory

event for mammalian sperm-oocyte interaction leading to fertilization requires further

studies to integrate the information regarding the role of the different signalling

pathways. These studies may provide the precise sequential role of the intracellular

messengers produced after the spermatozoa-ZP interaction has taken place. The

nature of the zona receptor (ZP2) in the acrosomal-reacted spermatozoa and the

acrosomal proteases in the development of the AR and penetration through the ZP

need to be clarified. Investigations of these and other related questions should

unravel the molecular processes involved in the acrosome reaction.


191

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